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Abstract:

This invention provides for the use of compounds represented by the
structure of the general formula (A):
##STR00001##
wherein L is a lipid or a phospholipid, Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol, Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms, X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000, wherein any
bond between L, Z, Y and X is either an amide or an esteric bond in
treating a subject suffering from a disease associated with elevated
level of a Matrix Metalloprotease (MMP) such as a malignant cancer.

Claims:

1. A method for treating a subject afflicted with lung cancer, comprising
the step of administering to said subject a composition comprising a
compound represented by the structure of the general formula (I):
##STR00038## wherein R1 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is glycosaminoglycan alginate or polygeline; and n is a
number from 1 to 1,000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, said spacer is directly linked to X via an amide or an
esteric bond and to said phosphatidylethanolamine via an amide bond.

2. The method of claim 1, wherein said n is a number from 2 to 1,000.

3. The method of claim 1, wherein said glycosaminoglycan is selected from
the group consisting of hyaluronic acid, heparin, heparan sulfate,
chondrotin sulfate, keratan, keratan sulfate, dermatan sulfate or a
derivative thereof.

4. The method of claim 1, wherein said phosphatidylethanolamine is a
myristoyl or palmitoyl phosphatidylethanolamine.

5. The method of claim 1, wherein said phosphatidylethanolamine is a
dipalmitoyl phosphatidylethanolamine, or dimyristoyl
phosphatidylethanolamine.

6. A method for attenuating invasiveness of a cancer cell, comprising the
step of subjecting said cancer cell to a composition comprising a
compound represented by the structure of the general formula (I):
##STR00039## wherein R1 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is glycosaminoglycan, alginate or polygeline; and n is a
number from 1 to 1,000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, said spacer is directly linked to X via an amide or an
esteric bond and to said phosphatidylethanolamine via an amide bond.

7. The method of claim 6, wherein said n is a number from 2 to 1,000.

8. The method of claim 6, wherein said glycosaminoglycan is selected from
the group consisting of hyaluronic acid, heparin, heparan sulfate,
chondrotin sulfate, keratan, keratan sulfate, dermatan sulfate or a
derivative thereof.

9. The method of claim 6, wherein said phosphatidylethanolamine is a
myristoyl or palmitoyl phosphatidylethanolamine.

10. The method of claim 6, wherein said phosphatidylethanolamine is a
dipalmitoyl phosphatidylethanolamine, or dimyristoyl
phosphatidylethanolamine.

11. A method for inhibiting proliferation of an endothelial cell,
comprising the step of subjecting said endothelial cell to a composition
comprising a compound represented by the structure of the general formula
(I): ##STR00040## wherein R1 is a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is glycosaminoglycan, alginate or polygeline; and n is a
number from 1 to 1,000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, said spacer is directly linked to X via an amide or an
esteric bond and to said phosphatidylethanolamine via an amide bond.

12. The method of claim 11, wherein said n is a number from 2 to 1,000.

13. The method of claim 11, wherein said glycosaminoglycan is selected
from the group consisting of hyaluronic acid, heparin, heparan sulfate,
chondrotin sulfate, keratan, keratan sulfate, dermatan sulfate or a
derivative thereof.

14. The method of claim 11, wherein said phosphatidylethanolamine is a
myristoyl or palmitoyl phosphatidylethanolamine.

15. The method of claim 11, wherein said phosphatidylethanolamine is a
dipalmitoyl phosphatidylethanolamine, or dimyristoyl
phosphatidylethanolamine.

16. The method of claim 11, wherein capillary formation is inhibited by
inhibiting proliferation of said endothelial cell.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. application Ser.
No. 12/463,792, filed May 11, 2009, which is a continuation-in-part of
U.S. application Ser. No. 11/822,423, filed Jul. 5, 2007 which is a
continuation-in-part of: (1) U.S. application Ser. No. 10/989,606 filed
Nov. 17, 2004, 2001, which is a continuation-in-part of U.S. application
Ser. No. 10/627,981, filed Jul. 28, 2003; and (2) U.S. application Ser.
No. 10/952,496 filed Sep. 29, 2004; each of which is a
continuation-in-part of U.S. application Ser. No. 09/756,765, filed Jan.
10, 2001, which claims priority to U.S. Provisional Application Ser. No.
60/174,907, filed Jan. 10, 2000 and U.S. Provisional Application Ser. No.
60/174,905, filed Jan. 10, 2000. Each and All patent applications
referenced above are hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] This invention is directed to lipid-GAG conjugates and
phospholipid-GAG conjugates for inhibiting a matrix metalloproteinase.

BACKGROUND OF THE INVENTION

[0003] Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, are
expressed in most colonic, gastric, and ovarian carcinomas, and they play
a key role in their invasiveness.

[0004] A major cause of morbidity in patients with cancer is the
metastatic spread of tumor cells, governed by a number of processes:
invasiveness of tumor cells through the basement membrane, proliferation
of the tumor cells in specific sites, and tumor vascularization which is
essential for its growth. The major components of the basement membrane,
comprising the barrier to the invading tumor cells, are collagen IV,
laminin and heparane sulfate proteoglycans. The degradation of
extracellular matrix (ECM) in mammalian cells is regulated by a family of
MMPs, including collagenases, gelatinases, stromelysins and membrane type
MMPs. The passage of tumor cells through the basement membrane begins
with the binding of the cell to laminin and subsequent activation of a
protease cascade, leading to the production of active MMPs from
pre-activated MMP forms or pre-<<Ps. These enzymes specifically
degrade the major structural element in the ECM: collagen IV. The
movement of cells across the basement membrane may occur in response to
specific chemotactic and motility factors produced by the host tissue.

[0005] MMP production and cancer cell invasiveness have been shown to
require the involvement of prostaglandins (PGs) and leukotrienes (LTs)
produced via the cyclooxygenases (COX) and lipoxygenases (LOX) pathways.
Both PGs and LTs are involved in the development of several types of
cancer in humans including: colon, breast, gastric and hepatocellular
carcinomas. Different eicosanoids have been shown to facilitate the
invasiveness of tumor cells, angiogenesis and tumor vascularization.

[0006] Lipid-conjugates having a pharmacological activity of inhibiting
the enzyme phospholipase A2 (PLA2, EC 3.1.1.4) are known in the prior
art. Phospholipase A2 catalyzes the breakdown of phospholipids at the
sn-2 position to produce a fatty acid and a lysophospholipid. The
activity of this enzyme has been correlated with various cell functions,
particularly with the production of lipid mediators such as eicosanoid
production (prostaglandins, thromboxanes and leukotrienes), platelet
activating factor and lysophospholipids. Since their inception,
lipid-conjugates have been subjected to intensive laboratory
investigation in order to obtain a wider scope of protection of cells and
organisms from injurious agents and pathogenic processes.

SUMMARY OF THE INVENTION

[0007] In one embodiment, provided a method for treating a subject
afflicted with a disease in which increased production of a matrix
metalloprotease (MMP) is associated with said disease, comprising the
step of administering to said subject a composition comprising a compound
represented by the structure of the general formula (A):

##STR00002##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol; Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
treating a subject afflicted with a disease in which increased production
of MMP is implicated.

[0008] In another embodiment, further provided is a method of treating a
subject afflicted with a metastatic cancer, comprising the step of
administering to said subject a composition comprising a compound
represented by the structure of the general formula (A):

##STR00003##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol; Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
treating a subject afflicted with a metastatic cancer.

[0009] In another embodiment, further provided is a method of inhibiting
the production of a matrix metalloprotease (MMP) in a cell, comprising
contacting said cell with a composition comprising a compound represented
by the structure of the general formula (A):

##STR00004##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol: Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
inhibiting invasiveness of a cancer cell.

[0010] In another embodiment, further provided is a method of treating a
subject afflicted with melanoma, comprising the step of administering to
the subject a composition comprising a compound represented by the
structure of the general formula (A):

##STR00005##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol; Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
treating a subject afflicted with melanoma.

[0011] In another embodiment, further provided is a method of inhibiting
invasiveness of a cancer cell, comprising the step of contacting said
cell with a composition comprising a compound represented by the
structure of the general formula (A):

##STR00006##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol: Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
inhibiting invasiveness of a cancer cell.

[0012] In another embodiment, further provided is a method of inhibiting a
collagenolytic activity of a cell, comprising the step of contacting said
cell with a composition comprising a compound represented by the
structure of the general formula (A):

##STR00007##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol; Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
inhibiting a collagenolytic activity of a Matrix metalloproteinase.

[0013] In another embodiment, further provided is a method A method of
inhibiting the production of a Matrix Metalloproteinase (MMP) in a cancer
cell, comprising the step of contacting said cell with a composition
comprising a compound represented by the structure of the general formula
(A):

##STR00008##

wherein L is a lipid or a phospholipid, Z is either nothing,
ethanolamine, serine, inositol, choline, or glycerol; Y is either nothing
or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer, wherein
X is a glycosaminoglycan; and n is a number from 2 to 1000; wherein any
bond between L, Z, Y and X is either an amide or an esteric bond, thereby
inhibiting the production of a Matrix Metalloproteinase (MMP) in a cancer
cell.

[0014] In one embodiment, further provided is a method of treating a
subject afflicted with lung cancer, comprising the step of administering
to the subject a composition comprising a compound represented by the
structure of the general formula (I):

##STR00009##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is glycosaminoglycan alginate or polygeline; and n is a
number from 1 to 1,000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, said spacer is directly linked to X via an amide or an
esteric bond and to said phosphatidylethanolamine via an amide bond.

[0015] In one embodiment, further provided is a method of attenuating
invasiveness of a cancer cell, comprising the step of subjecting the
cancer cell to a composition comprising a compound represented by the
structure of the general formula (I):

##STR00010##

wherein is R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is glycosaminoglycan alginate or polygeline; and n is a
number from 1 to 1,000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, said spacer is directly linked to X via an amide or an
esteric bond and to said phosphatidylethanolamine via an amide bond.

[0016] In one embodiment, further provided is a method of inhibiting
proliferation of an endothelial cell or inhibiting capillary formation,
comprising the step of subjecting the endothelial cell to a composition
comprising a compound represented by the structure of the general formula
(I):

##STR00011##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is glycosaminoglycan alginate or polygeline; and n is a
number from 1 to 1,000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, said spacer is directly linked to X via an amide or an
esteric bond and to said phosphatidylethanolamine via an amide bond.

[0017] According to one embodiment, n is a number from 1 to 1,000. In
another embodiment, n is a number from 2 to 1,000. In another embodiment,
n is a number from 2 to 500. In another embodiment, n is a number from 1
to 500. In another embodiment, n is a number from 1 to 100. In another
embodiment, n is a number from 2 to 1000. In another embodiment, n is a
number from 2 to 100. In another embodiment, n is a number from 2 to 200.
In another embodiment, n is a number from 3 to 300. In another
embodiment, n is a number from 10 to 400. In another embodiment, n is a
number from 50 to 500. In another embodiment, n is a number from 100 to
300. In another embodiment, n is a number from 300 to 500. In another
embodiment, n is a number from 500 to 800. In another embodiment, n is a
number from 500 to 1000.

[0018] According to one embodiment, the glycosaminoglycan is selected from
the group consisting of hyaluronic acid, heparin, heparan sulfate,
chondrotin sulfate, keratan, keratan sulfate, dermatan sulfate or a
derivative thereof.

[0019] The phosphatidylethanolamine may be a myristoyl or palmitoyl
phosphatidylethanolamine, or further dipalmitoyl
phosphatidylethanolamine, or dimyristoyl phosphatidylethanolamine.

BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1 is a bar graph showing the inhibitory effect of ExPLI (lipid
conjugates) on the invasion capacity of HT-1080 cell. HT-1080 cells were
treated with the ExPLI HyPE, composed of Hyaluronic acid (HA) conjugated
PE and with HA, at the indicated concentrations, for 24 h, than washed
and placed on a Matrigel membrane. Cell invasion through the Matrigel was
determined. Each datum is Mean and SD for 3 replications (a, b,
P<0.05).

[0021]FIG. 2 upper panel is a micrograph of a gel followed by a bar graph
(lower panel). This graph demonstrates the inhibitory effect of HyPE on
MMP-2 and MMP-9 activity. HT-1080 were incubated for 24 h with either
HyPE or HA. The cultured medium was then collected and subjected to
determination of MMP-2 (72 kDa) and MMP-9 (96 kDa) content and their
collagenolytic activity, using zymography as described in Materials and
methods. Each datum is Mean and SD for 4 replications (*, P<0.05, **,
P<0.01).

[0022] FIG. 3 are bar graphs showing the inhibitory effect of ExPLI
((lipid conjugates) on PLA2 activity in HT-1080 cells as evidenced by the
release of Arachidonic acid (AA) (lower panel) or Oleic acid (upper
panel) from HT-1080 cells. HT-1080 cells were metabolically labeled by
overnight incubation with either 3H-arachidonic acid or 3H-oleic acid,
then washed and the release of the labeled AA or OA into the culture
medium during the indicated time, in the absence (clear bar) or presence
(black bar) of HyPE was measured. Each datum is Mean and SD for 3
replications. (*, P<0.05).

[0024] FIG. 5 is a bar graph showing the effect of heat inactivation on
lipolytic activity of porcine pancreatic and crotalus atrox sPLA2s
(ppPLA2 and caPLA2, respectively). The enzymes were denatured by heating
at 95° C. for 15 min, and their lipolytic capacity was determined
by their ability to hydrolyze 4N3OBA, as in the experimental section.
Each datum is Mean and SD for 3 replications. (*, P<0.05).

[0025] FIG. 6 are graphs followed by gel micrographs demonstrating the
effect of heat inactivation of ppPLA2 on its ability to induce MMP
activity/production. HT-1080 cells were treated with either intact or
denatured (d) ppPLA2 for 6 h, and the activity of MMP-9 (A) and MMP-2 (B)
activity was determined by zymography. Each datum is Mean and SD for 3
replications (*, P<0.01).

[0026] FIG. 7 presents cPLA2 phosphorylation by ppPLA2 and its suppression
by heat inactivation or sPLA2 inhibitor. HT-1080 cells were treated with
ppPLA2 in the absence or presence of HyPE, or with denatured ppPLA2 for
15 min prior to protein isolation. The extent of cPLA2 phosphorylation
was determined by Western blot analysis with specific antibodies directed
against cPLA2 phosphorylated on Ser505 and with specific antibody
directed against the total (phosphorylated and non-phosphorylated) cPLA2.
Each datum is Mean and SD for 2 replications (a, b, P<0.05).

[0027] FIG. 8 is a bar graph showing the effect of HyPE on the
transcription of sPLA2 Types IIA and IB by HT-1080 cells. HT-1080 cells
were treated for 24 h in the absence or presence of HyPE (10 microM)
prior to RNA extraction. The transcription of sPLA2-IB and IIA in these
cells were analyzed by RT-PCR using the primers as described in the
experimental section. Sample loading was verified by 28s expression. Each
datum is Mean and SD for 3 replications (*, P<0.05).

[0041] FIG. 17 is an HPLC chromatogram of HyPE prepared according to
Example 10.

[0042] FIG. 18 depicts a conceptual diagram of the reaction vessel
features required to practice the methods of this invention.

[0043]FIG. 19: depicts a chromatogram of the HyPE reaction from Example
11 after 6 hours.

[0044]FIG. 20: depicts the GPC analysis of final HyPE isolated from
Example 11.

[0045] It will be appreciated that for simplicity and clarity of
illustration, elements shown in the figures have not necessarily been
drawn to scale. For example, the dimensions of some of the elements may
be exaggerated relative to other elements for clarity. Further, where
considered appropriate, reference numerals may be repeated among the
figures to indicate corresponding or analogous elements.

DETAILED DESCRIPTION OF THE INVENTION

[0046] In one embodiment, this invention provides a method of inhibiting a
Matrix metalloproteinase (MMP). In another embodiment, the invention
provides a method of inhibiting MMP in a subject. In another embodiment,
the invention provides a method of inhibiting MMP in a cancerous cell. In
another embodiment, the invention provides a method of inhibiting MMP in
a cancerous cell in a subject. In another embodiment, the invention
provides a method of inhibiting MMP in a metastatic cell. In another
embodiment, the invention provides a method of inhibiting MMP in a
metastatic cell in a subject. In another embodiment, the invention
provides a method of inhibiting MMP in a tumor cell. In another
embodiment, the invention provides a method of inhibiting MMP in a tumor
cell in a subject.

[0047] In another embodiment, the invention provides a method based on the
use of the compounds of invention as MMP inhibitors. In another
embodiment, the invention provides a method based on the use of the
compounds of invention as MMP 2 inhibitors. In another embodiment, the
invention provides a method based on the use of the compounds of
invention as MMP 9 inhibitors.

[0048] In another embodiment, the invention provides a method of
inhibiting the development of a primary tumor or a lesion to a metastatic
cancer.

[0049] In another embodiment, this invention provides a method for
treating a subject afflicted with a disease or a pathology characterized
by elevated MMP levels via administration of a compound comprising a
lipid or a phospholipid bonded, directly or via a spacer group, to a
physiologically acceptable monomer, dimer, oligomer, or polymer. In
another embodiment, this invention provides a method for treating a
subject afflicted with a disease or a pathology mediated by elevated MMP
levels via administration of a compound comprising a lipid or a
phospholipid bonded, directly or via a spacer group, to a physiologically
acceptable monomer, dimer, oligomer, or polymer. In another embodiment,
this invention provides a method for treating a subject afflicted with a
disease or a pathology induced by elevated MMP levels via administration
of a compound comprising a lipid or a phospholipid bonded, directly or
via a spacer group, to a physiologically acceptable monomer, dimer,
oligomer, or polymer. In another embodiment, this invention provides that
a compound comprising a lipid or a phospholipid bonded, directly or via a
spacer group, to a physiologically acceptable monomer, dimer, oligomer,
or polymer is a MMP inhibitor. In another embodiment, this invention
provides a method for treating a subject afflicted with a malignant tumor
via administration of a compound comprising a lipid or a phospholipid
bonded, directly or via a spacer group, to a physiologically acceptable
monomer, dimer, oligomer, or polymer. In another embodiment, this
invention provides a method for inhibiting cancer spread in a subject via
administration of a compound comprising a lipid or a phospholipid bonded,
directly or via a spacer group, by an amide or an ester bond to a
glycosaminoglycan.

[0050] In another embodiment, this invention provides administration of
the conjugates for the treatment of diseases which require controlling
phospholipase A2 activities, controlling the production and/or action of
lipid mediators, amelioration of damage to cell surface by
glycosaminoglycans (GAG) and proteoglycans, controlling the production of
oxygen radicals and nitric oxide, protection of lipoproteins from
damaging agents, anti-oxidant therapy; anti-endotoxin therapy;
controlling of cytokine, chemokine and interleukin production;
controlling the proliferation of cells, controlling of angiogenesis and
organ vascularization; inhibition of invasion-promoting enzymes,
inhibition of a MMP, controlling of cell invasion, controlling of
leukocyte activation, adhesion and extravasation, amelioration of
ischemia/reperfusion injury, inhibition of lymphocyte activation,
controlling of blood vessel and airway contraction, protection of blood
brain barrier, controlling of neurotransmitter production and action or
extracorporeal tissue preservation.

[0051] In another embodiment of the invention, the lipid-conjugates
described are used in a process for manufacture of a composition for the
treatment of diseases which requires controlling phospholipase A2
activities, controlling the production and/or action of lipid mediators,
amelioration of damage to cell surface by glycosaminoglycans (GAG) and
proteoglycans, controlling of cytokine, chemokine and interleukine
production; controlling the proliferation of cells, inhibiting MMP
activity/production (expression and/or transcription) controlling of
angiogenesis and organ vascularization; inhibition of invasion-promoting
enzymes, controlling of cell invasion, controlling of white cell
activation, adhesion and extravasation.

[0052] Metastasis, the spread of cancer cells to ectopic sites, is
frequently a vasculature dependent process as well, often referred to as
hematogenous spread. The physiological barrier imposed by the blood
vessel wall, comprised from elements such as endothelial cells and
basement membrane substance, is normally highly selective to the passage
of cells. However, metastatic cells abrogate this barrier, employing a
variety of mechanisms, some of which have been established in the
scientific literature. For example, such abnormal cells produce
hydrolytic enzymes which degrade the extracellular matrix and associated
components of the vascular barrier, such as collagenase, heparinase, and
hyaluronidase. Thus a critical factor in the metastatic process is the
ability of cancer cells to intrude through or permeate the wall of the
blood vessel lumen, thus arriving to invade a new tissue site after
travel through the circulation. In another embodiment, a MMP inhibitor as
described herein inhibits the intruding capacity of cells. In another
embodiment, a MMP inhibitor as described herein inhibits the intruding
capacity of metastatic cells. In another embodiment, a MMP inhibitor as
described herein inhibits the intruding capacity of tumor cells.

[0054] In another embodiment, the treatment requires protection of
lipoproteins from damaging agents. In another embodiment, the treatment
requires controlling the proliferation of cells. In another embodiment,
the treatment requires controlling of angiogenesis and organ
vascularization. In another embodiment, the treatment requires inhibition
of invasion-promoting enzymes. In another embodiment, the treatment
requires controlling of cell invasion. In another embodiment, the
invading cells are white blood cells. In another embodiment, the invading
cells are cancer cells. In another embodiment, the treatment requires
controlling of white cell activation, adhesion or extravasation. In
another embodiment, the treatment requires amelioration of ischemia or
reperfusion injury. In another embodiment, the treatment requires
inhibition of lymphocyte activation. In another embodiment, the treatment
requires protection of blood brain barrier. In another embodiment, the
treatment requires control of neurotransmitter production and action. In
another embodiment, the treatment requires controlling of blood vessel
and airway contraction. In another embodiment, the treatment requires
extracorporeal tissue preservation.

[0055] In one embodiment, the invention provides a method of treating a
subject afflicted with a disease, wherein the treatment of the disease
requires controlling phospholipase A2 activities; controlling the
production and/or action of lipid mediators, such as eicosanoids,
platelet activating factor (PAF) and lyso-phospholipids; amelioration of
damage to cell surface glycosaminoglycans (GAG) and proteoglycans;
controlling the production of oxygen radicals and nitric oxide;
protection of cells, tissues, and plasma lipoproteins from damaging
agents, such as reactive oxygen species (ROS) and phospholipases;
anti-oxidant therapy; anti-endotoxin therapy; controlling of cytokine,
chemokine and interleukine production; controlling the proliferation of
cells, including smooth muscle cells, endothelial cells and skin
fibroblasts; controlling of angiogenesis and organ vascularization;
inhibition of invasion-promoting enzymes, such as collagenase,
heparinase, heparanase and hyaluronidase; controlling of cell invasion;
controlling of white cell activation, adhesion and extravasation;
amelioration of ischemia/reperfusion injury, inhibition of lymphocyte
activation; controlling of blood vessel and airway contraction;
protection of blood brain barrier; controlling of neurotransmitter (e.g.,
dopamine) production and action (e.g., acethylcholine); extracorporeal
tissue preservation or any combination thereof.

Compounds

[0056] In one embodiment, reference to a compound for use in a method of
the present invention refers to one comprising a lipid or phospholipid
moiety bound to a physiologically acceptable monomer, dimer, oligomer, or
polymer. In one embodiment, the compounds for use in the present
invention are referred to as "Lipid-conjugates." In another embodiment,
reference to a MMP inhibitor for use in a method of the present invention
refers to one comprising a lipid or phospholipid moiety bound to a
physiologically acceptable monomer, dimer, oligomer, or polymer. In one
embodiment, the compounds for use in the present invention are referred
to as "Lipid-conjugates." In another embodiment, compounds for use in the
present invention are described by the general formula:

[phosphatidylethanolamine-Y]n-X [phosphatidylserine-Y]n-X
[phosphatidylcholine-Y]n-X [phosphatidylinositol-Y]n-X
[phosphatidylglycerol-Y]n-X [phosphatidic acid-Y]n-X
[lyso-phospholipid-Y]n-X [diacyl-glycerol-Y]n-X [monoacyl-glycerol-Y]n-X
[sphingomyelin-Y]n-X [sphingosine-Y]n-X [ceramide-Y]n-X wherein Y is
either nothing or a spacer group ranging in length from 2 to 30 atoms;
and X is a physiologically acceptable monomer, dimer, oligomer or
polymer: and n is the number of lipid molecules bound to a molecule of X,
wherein n is a number from 1 to 1000 or a number from 2 to 1000.

[0057] In one embodiment, the invention provides low-molecular weight
Lipid-conjugates, which possess pharmacological activity, which are
characterized by the general formula described hereinabove.

[0058] In one embodiment of the invention, the physiologically acceptable
monomer is salicylate. In another embodiment, the physiologically
acceptable monomer is salicylic acid. In another embodiment, the
physiologically acceptable monomer is acetyl salicylic acid. In another
embodiment, the physiologically acceptable monomer is aspirin. In another
embodiment, the physiologically acceptable monomer is a monosaccharide.
In another embodiment, the physiologically acceptable monomer is
lactobionic acid. In another embodiment, the physiologically acceptable
monomer is glucoronic acid. In another embodiment, the physiologically
acceptable monomer is maltose. In another embodiment, the physiologically
acceptable monomer is an amino acid. In another embodiment, the
physiologically acceptable monomer is glycine. In another embodiment, the
physiologically acceptable monomer is a carboxylic acid. In another
embodiment, the physiologically acceptable monomer is an acetic acid. In
another embodiment, the physiologically acceptable monomer is a butyric
acid. In another embodiment, the physiologically acceptable monomer is a
dicarboxylic acid. In another embodiment, the physiologically acceptable
monomer is a fatty acid. In another embodiment, the physiologically
acceptable monomer is a dicarboxylic fatty acid. In another embodiment,
the physiologically acceptable monomer is a glutaric acid. In another
embodiment, the physiologically acceptable monomer is succinic acid. In
another embodiment, the physiologically acceptable monomer is dodecanoic
acid. In another embodiment, the physiologically acceptable monomer is
didodecanoic acid. In another embodiment, the physiologically acceptable
monomer is bile acid. In another embodiment, the physiologically
acceptable monomer is cholic acid. In another embodiment, the
physiologically acceptable monomer is cholesterylhemisuccinate.

[0059] In one embodiment of the invention, the physiologically acceptable
dimer or oligomer is a dipeptide. In another embodiment, the
physiologically acceptable dimer or oligomer is a disaccharide. In
another embodiment, the physiologically acceptable dimer or oligomer is a
trisaccharide. In another embodiment, the physiologically acceptable
dimer or oligomer is an oligosaccharide. In another embodiment, the
physiologically acceptable dimer or oligomer is an oligopeptide. In
another embodiment, the physiologically acceptable dimer or oligomer is a
glycoprotein mixture. In another embodiment, the physiologically
acceptable dimer or oligomer is a di- or trisaccharide monomer unit of a
polysaccharide. In another embodiment, the physiologically acceptable
dimer or oligomer is a di- or trisaccharide monomer unit of a
polypyranose. In another embodiment, the physiologically acceptable dimer
or oligomer is a di- or trisaccharide monomer unit of a
glycosaminogylcan. In another embodiment, the physiologically acceptable
dimer or oligomer is a di- or trisaccharide monomer unit of a hyaluronic
acid. In another embodiment, the physiologically acceptable dimer or
oligomer is a di- or trisaccharide monomer unit of a heparin. In another
embodiment, the physiologically acceptable dimer or oligomer is a di- or
trisaccharide monomer unit of a heparan sulfate. In another embodiment,
the physiologically acceptable dimer or oligomer is a di- or
trisaccharide monomer unit of a keratin. In another embodiment, the
physiologically acceptable dimer or oligomer is a di- or trisaccharide
monomer unit of a keratan sulfate. In another embodiment, the
physiologically acceptable dimer or oligomer is a di- or trisaccharide
monomer unit of a chondroitin. In another embodiment, the chondroitin is
chondoitin sulfate. In another embodiment, the chondroitin is
chondoitin-4-sulfate. In another embodiment, the chondroitin is
chondoitin-6-sulfate. In another embodiment, the physiologically
acceptable dimer or oligomer is a di- or trisaccharide monomer unit of a
dermatin. In another embodiment, the physiologically acceptable dimer or
oligomer is a di- or trisaccharide monomer unit of a dermatan sulfate. In
another embodiment, the physiologically acceptable dimer or oligomer is
dextran. In another embodiment, the physiologically acceptable dimer or
oligomer is polygeline (`Haemaccel`). In another embodiment, the
physiologically acceptable dimer or oligomer is alginate. In another
embodiment, the physiologically acceptable dimer or oligomer is
hydroxyethyl starch (Hetastarch). In another embodiment, the
physiologically acceptable dimer or oligomer is ethylene glycol. In
another embodiment, the physiologically acceptable dimer or oligomer is
carboxylated ethylene glycol.

[0060] In one embodiment, the physiologically acceptable polymer is a
polysaccharide. In another embodiment, the physiologically acceptable
polymer is a homo-polysaccharide. In another embodiment, the
physiologically acceptable polymer is a hetero-polysaccharide. In another
embodiment, the physiologically acceptable polymer is a polypyranose. In
another embodiment of the invention, the physiologically acceptable
polymer is a glycosaminoglycan. In another embodiment, the
physiologically acceptable polymer is hyaluronic acid. In another
embodiment, the physiologically acceptable polymer is heparin. In another
embodiment, the physiologically acceptable polymer is heparan sulfate. In
another embodiment, the physiologically acceptable polymer is
chondroitin. In another embodiment, the chondroitin is
chondoitin-4-sulfate. In another embodiment, the chondroitin is
chondoitin-6-sulfate. In another embodiment, the physiologically
acceptable polymer is keratin. In another embodiment, the physiologically
acceptable polymer is keratan sulfate. In another embodiment, the
physiologically acceptable polymer is dermatin. In another embodiment,
the physiologically acceptable polymer is dermatan sulfate. In another
embodiment, the physiologically acceptable polymer is
carboxymethylcellulose. In another embodiment, the physiologically
acceptable polymer is dextran. In another embodiment, the physiologically
acceptable polymer is polygeline (`Haemaccel`). In another embodiment,
the physiologically acceptable polymer is alginate. In another
embodiment, the physiologically acceptable polymer is hydroxyethyl starch
(`Hetastarch`). In another embodiment, the physiologically acceptable
polymer is polyethylene glycol. In another embodiment, the
physiologically acceptable polymer is polycarboxylated polyethylene
glycol. In another embodiment, the physiologically acceptable polymer is
a peptide. In another embodiment, the physiologically acceptable polymer
is an oligopeptide. In another embodiment, the physiologically acceptable
polymer is a polyglycan. In another embodiment, the physiologically
acceptable polymer is a protein. In another embodiment, the
physiologically acceptable polymer is a glycoprotein mixture.

[0061] The following terms may be used according to certain embodiments of
the invention: phosphatidylethanolamine (PE) linked to
carboxymethylcellulose (referred to as CMPE), to hyaluronic acid
(referred to as HYPE), to heparin (referred to as HepPE), to chondroitin
sulfate A (referred to as CSAPE), to Polygeline (haemaccel) (referred to
HemPE) or to hydroxyethylstarch (referred to as HesPE).
Phosphatidylserine (PS) and other phospholipids linked conjugates may be
named similarly.

[0063] In one embodiment of the invention, the lipid or phospholipid
moiety is phosphatidic acid. In another embodiment, lipid or phospholipid
moiety is an acyl glycerol. In another embodiment, lipid or phospholipid
moiety is monoacylglycerol. In another embodiment, lipid or phospholipid
moiety is diacylglycerol. In another embodiment, lipid or phospholipid
moiety is triacylglycerol. In another embodiment, lipid or phospholipid
moiety is sphingosine. In another embodiment, lipid or phospholipid
moiety is sphingomyelin. In another embodiment, lipid or phospholipid
moiety is ceramide. In another embodiment, lipid or phospholipid moiety
is phosphatidylethanolamine. In another embodiment, lipid or phospholipid
moiety is phosphatidylserine. In another embodiment, lipid or
phospholipid moiety is phosphatidylcholine. In another embodiment, lipid
or phospholipid moiety is phosphatidylinositol. In another embodiment,
lipid or phospholipid moiety is phosphatidylglycerol. In another
embodiment, lipid or phospholipid moiety is an ether or alkyl
phospholipid derivative thereof.

[0064] In one embodiment, the set of compounds comprising
phosphatidylethanolamine covalently bound to a physiologically acceptable
monomer, dimmer, oligomer, or polymer, is referred to herein as the
PE-conjugates. In one embodiment, the phosphatidylethanolamine moiety is
dipalmitoyl phosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is dimyristoyl phosphatidylethanolamine.
In another embodiment, related derivatives, in which either
phosphatidylserine, phosphatidylcholine, phosphatidylinositol,
phosphatidic acid or phosphatidylglycerol are employed in lieu of
phosphatidylethanolamine as the lipid moiety provide equivalent
therapeutic results, based upon the biological experiments described
below for the Lipid-conjugates and the structural similarities shared by
these compounds.

[0065] As defined by the structural formulae provided herein for the
Lipid-conjugates or phospholipids-conjugates, these compounds may contain
between one to one thousand lipid or phospholipid moieties bound to a
single physiologically acceptable polymer molecule. In one embodiment of
this invention, n is a number from 1 to 1000. In another embodiment, n is
a number from 2 to 500. In another embodiment, n is a number from 1 to
500. In another embodiment, n is a number from 1 to 100. In another
embodiment, n is a number from 2 to 1000. In another embodiment, n is a
number from 2 to 100. In another embodiment, n is a number from 2 to 200.
In another embodiment, n is a number from 3 to 300. In another
embodiment, n is a number from 10 to 400. In another embodiment, n is a
number from 50 to 500. In another embodiment, n is a number from 100 to
300. In another embodiment, n is a number from 300 to 500. In another
embodiment, n is a number from 500 to 800. In another embodiment, n is a
number from 500 to 1000.

[0066] In one embodiment of the invention, when the conjugated moiety is a
polymer, the ratio of lipid moieties covalently bound may range from one
to one thousand lipid or phospholipids (PL) residues per polymer
molecule, depending upon the nature of the polymer and the reaction
conditions employed. For example, the relative quantities of the starting
materials, or the extent of the reaction time, may be modified in order
to obtain Lipid-conjugate or Phospholipid (PL)-conjugate products with
either high or low ratios of lipid residues per polymer, as desired.

[0067] In the methods, according to embodiments of the invention, the
Lipid-conjugates or Phospholipid-conjugate administered to a subject are
comprised of at least one lipid or phospholipid moiety covalently bound
through an atom of the polar head group to a monomeric or polymeric
moiety (referred to herein as the conjugated moiety) of either low or
high molecular weight. In one embodiment, the conjugated moiety is
conjugated to the lipid, phospholipid, or spacer via an ester bond. In
another embodiment, the conjugated moiety is conjugated to the lipid,
phospholipid, or spacer via an amide bond.

[0068] When desired, an optional bridging moiety can be used to link the
lipid or phospholipid moiety to the monomer or polymeric moiety. The
composition of some phospholipid-conjugates of high molecular weight, and
associated analogues, are the subject of U.S. Pat. No. 5,064,817, which
is incorporated herein in its entirety by reference.

[0069] In one embodiment, the term "moiety" means a chemical entity
otherwise corresponding to a chemical compound, which has a valence
satisfied by a covalent bond.

[0070] In some cases, according to embodiments of the invention, the
monomer or polymer chosen for preparation of the Lipid-conjugate or
Phospholipid-conjugate may in itself have selected biological properties.
For example, both heparin and hyaluronic acid are materials with known
physiological functions. In the present invention, however, the
Lipid-conjugates or Phospholipid-conjugate formed from these substances
as starting materials display a new and wider set of pharmaceutical
activities than would be predicted from administration of either heparin
or hyaluronic acid which have not been bound by covalent linkage to a
phospholipid. It can be shown, by standard comparative experiments that
phosphatidylethanolamine (PE) linked to hyaluronic acid (Compound XXII),
to heparin (Compound XXIV), to chondroitin sulfate A (Compound XXV), to
carboxymethylcellulose (Compound XXVI), to Polygeline (haemaccel)
(Compound XXVII), or to hydroxyethylstarch (Compound XXVIII), are far
superior in terms of potency and range of useful pharmaceutical activity
to the free conjugates (the polymers above and the like). In fact, these
latter substances are, in general, not considered useful in methods for
inhibiting MMP activity or production in a cell. Thus, the combination of
a phospholipid such as phosphatidylethanolamine, or related phospholipids
which differ with regard to the polar head group, such as
phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylinositol
(PI), and phosphatidylglycerol (PG), results in the formation of a
compound which has novel pharmacological properties when compared to the
starting materials alone. In the cases described herein, the diversity of
biological activities and the effectiveness in disease exhibited by the
compounds far exceed the properties anticipated by use of the starting
materials themselves, when administered alone or in combination.

[0071] The biologically active Lipid-conjugates or Phospholipid-conjugates
described herein can have a wide range of molecular weights, e.g., above
50,000 (up to a few hundred thousands) when it is desirable to retain the
conjugates in the vascular system and below 50,000 when targeting to
extravascular systems is desirable. The sole limitation on the molecular
weight and the chemical structure of the conjugated moiety is that it
does not result in a Lipid-conjugate or Phospholipid-conjugate devoid of
the desired biological activity, or lead to chemical or physiological
instability to the extent that the Lipid-conjugate or
Phospholipid-conjugate is rendered useless as a drug in the method of use
described herein.

[0072] In one embodiment, the compound for use in the present invention is
represented by the structure of the general formula (A):

##STR00012##

wherein L is a lipid or a phospholipid; Z is either nothing,
ethanolamine, serine, inositol, choline, phosphate, or glycerol; Y is
either nothing or a spacer group ranging in length from 2 to 30 atoms; X
is a physiologically acceptable monomer, dimer, oligomer, or polymer; and
n is a number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between L, Z, Y and X is either an amide or an esteric bond.

[0073] In one embodiment, L of Compound A is phospholipids (PL). In
another embodiment, L of Compound A is a lipid.

[0074] In one embodiment, L is phosphatidyl, Z is ethanolamine, wherein L
and Z are chemically bonded resulting in phosphatidylethanolamine, Y is
nothing, and X is carboxymethylcellulose. In another embodiment, L is
phosphatidyl, Z is ethanolamine, wherein L and Z are chemically bonded
resulting in phosphatidylethanolamine, Y is nothing, and X is a
glycosaminoglycan. In one embodiment, the phosphatidylethanolamine moiety
is dipalmitoyl phosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is dimyristoyl phosphatidylethanolamine.
In another embodiment, the phosphatidylethanolamine moiety is
1-Acyl-2-Acyl-sn-Glycero-3-Phosphoethanolamine. In another embodiment,
the phosphatidylethanolamine moiety is
1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is
1-hexadecanoyl-2-[(Z)-octadec-9-enoyl]-sn-glycero-3-phospho}ethanolamine.
In another embodiment, the phosphatidylethanolamine moiety is
1,2-distearoylphosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is 1,2-distearoylphosphatidylethanolamine
zwitterions. In another embodiment, the phosphatidylethanolamine moiety
is 1,2-distearoylphosphatidylethanolaminium. In another embodiment, the
phosphatidylethanolamine moiety is phosphatidyldi-N-methylethanolamines.
In another embodiment, the phosphatidylethanolamine moiety is
phosphatidyl-N-methylethanolamines.

[0075] In another embodiment, the phosphatidylethanolamine moiety is a
transesterified phosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is dipalmitoyl phosphatidylethanolamine.
In another embodiment, the phosphatidylethanolamine moiety is palmitoyl
oleoyl phosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is dioleoyl phosphatidylethanolamine. In
another embodiment, the phosphatidylethanolamine moiety is a PE
conjugated to a moiety selected from the group comprising of dicarboxylic
acids, polyethylene glycols, polyalkyl ethers and gangliosides.

[0076] In another embodiment, the phosphatidylethanolamine moiety is a
synthetic analogs of phosphatidylethanolamine. In another embodiment, the
phosphatidylethanolamine moiety is isolated from natural sources. In
another embodiment, the phosphatidylethanolamine moiety is synthesized
according to established chemical procedures, or enzymatically
synthesized using the corresponding phosphatidyl choline compound in the
presence of ethanolamine and phospholipase D.

[0077] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (I):

##STR00013##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; and X is either a physiologically acceptable monomer, dimer,
oligomer or a physiologically acceptable polymer; and n is a number from
1 to 1,000 or 2 to 1000; wherein if Y is nothing the
phosphatidylethanolamine is directly linked to X via an amide bond and if
Y is a spacer, the spacer is directly linked to X via an amide or an
esteric bond and to the phosphatidylethanolamine via an amide bond.

[0078] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (I),
wherein X is glycosaminoglycan (GAG). In another embodiment the compound
for use is represented by the structure of formula I, wherein X is
glycosaminoglycan (GAG) and n is a number from 1 to 70. In another
embodiment, the molecular weight of said GAG is between 5 to 20 kD

[0079] Examples of phosphatidylethanolamine (PE) moieties are analogues of
the phospholipid in which the chain length of the two fatty acid groups
attached to the glycerol backbone of the phospholipid varies from 2-30
carbon atoms length, and in which these fatty acids chains contain
saturated and/or unsaturated carbon atoms. In lieu of fatty acid chains,
alkyl chains attached directly or via an ether linkage to the glycerol
backbone of the phospholipid are included as analogues of PE. In one
embodiment, the PE moiety is dipalmitoyl-phosphatidyl-ethanolamine. In
another embodiment, the PE moiety is
dimyristoyl-phosphatidyl-ethanolamine.

[0080] Phosphatidyl-ethanolamine and its analogues may be from various
sources, including natural, synthetic, and semisynthetic derivatives and
their isomers.

[0081] Phospholipids which can be employed in lieu of the PE moiety are
N-methyl-PE derivatives and their analogues, linked through the amino
group of the N-methyl-PE by a covalent bond; N,N-dimethyl-PE derivatives
and their analogues linked through the amino group of the N,N-dimethyl-PE
by a covalent bond.

[0082] For PE-conjugates and PS-conjugates, the phospholipid is linked to
the conjugated monomer or polymer moiety through the nitrogen atom of the
phospholipid polar head group, either directly or via a spacer group. For
PC, PI, and PG conjugates, the phospholipid is linked to the conjugated
monomer or polymer moiety through either the nitrogen or one of the
oxygen atoms of the polar head group, either directly or via a spacer
group.

[0083] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (II):

##STR00014##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is a physiologically acceptable monomer, dimer, oligomer or
polymer wherein X is a glycosaminoglycan; and n is a number from 1 to
1000 or a number from 2 to 1000; wherein if Y is nothing, the
phosphatidylserine is directly linked to X via an amide bond and if Y is
a spacer, the spacer is directly linked to X via an amide or an esteric
bond and to the phosphatidylserine via an amide bond.

[0084] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (II),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0085] In one embodiment, the phosphatidylserine may be bonded to Y, or to
X if Y is nothing, via the COO.sup.- moiety of the phosphatidylserine.

[0086] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (III):

##STR00015##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, inositol, choline, or glycerol; Y is either
nothing or a spacer group ranging in length from 2 to 30 atoms; X is a
physiologically acceptable monomer, dimer, oligomer, or polymer wherein X
is a glycosaminoglycan; and n is a number from 1 to 1000 or a number from
2 to 1000; wherein any bond between the phosphatidyl, Z, Y and X is
either an amide or an esteric bond.

[0087] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (III),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0088] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IV):

##STR00016##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, inositol, choline, ethanolamine, serine or
glycerol; Y is either nothing or a spacer group ranging in length from 2
to 30 atoms; X is a physiologically acceptable monomer, dimer, oligomer,
or polymer wherein X is a glycosaminoglycan; and n is a number from 1 to
1000 or a number from 2 to 1000; wherein any bond between the
phospholipid, Z, Y and X is either an amide or an esteric bond.

[0089] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IV),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD

[0090] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (V):

##STR00017##

wherein to R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, inositol, choline,
ethanolamine, serine or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan;
and n is a number from 1 to 1000 or a number from 2 to 1000; wherein any
bond between the phospholipid, Z, Y and X is either an amide or an
esteric bond.

[0091] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (V),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0092] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (VI):

##STR00018##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, inositol, choline, ethanolamine, serine or
glycerol; Y is either nothing or a spacer group ranging in length from 2
to 30 atoms; X is a physiologically acceptable monomer, dimer, oligomer,
or polymer wherein X is a glycosaminoglycan; and n is a number from 1 to
1000 or a number from 2 to 1000; wherein any bond between the
phospholipid, Z, Y and X is either an amide or an esteric bond.

[0093] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (VI),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0094] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (VII):

##STR00019##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, inositol, ethanolamine,
serine, choline, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan;
and n is a number from 1 to 1000 or a number from 2 to 1000; wherein any
bond between the phospholipid, Z, Y and X is either an amide or an
esteric bond.

[0095] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (VII),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0096] In one embodiment of the invention, the conjugate comprises
phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid
(PA), wherein Z is nothing, and phosphatidylglycerol (PG) as defined as
compounds of the general formula (III).

[0097] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (VIII):

##STR00020##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, ethanolamine, serine,
inositol, choline, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan;
and n is a number from 1 to 1000 or a number from 2 to 1000; wherein any
bond between the phospholipid, Z, Y and X is either an amide or an
esteric bond.

[0098] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (VIII),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0099] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IX):

##STR00021##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, ethanolamine, serine,
inositol, choline, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan;
and n is a number from 1 to 1000 or a number from 2 to 1000; wherein any
bond between the phospholipid, Z, Y and X is either an amide or an
esteric bond.

[0100] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IX),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0101] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IXa):

##STR00022##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, ethanolamine, serine,
inositol, choline, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan;
and n is a number from 1 to 1000 or a number from 2 to 1000; wherein any
bond between the phospholipid, Z, Y and X is either an amide or an
esteric bond.

[0102] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IXa),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0103] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IXb):

##STR00023##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, ethanolamine, serine,
inositol, choline, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer, or polymer wherein X is a glycosaminoglycan;
and n is a number from 1 to 1000 or a number from 2 to 1000; wherein any
bond between the phospholipid, Z, Y and X is either an amide or an
esteric bond.

[0104] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (IXb),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0105] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (X):

##STR00024##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; Y is either nothing or a spacer group ranging in length from 2
to 30 atoms; X is a physiologically acceptable monomer, dimer, oligomer,
or polymer wherein X is a glycosaminoglycan; and n is a number from 1 to
1000 or a number from 2 to 1000; wherein any bond between the ceramide
phosphoryl, Z, Y and X is either an amide or an esteric bond.

[0106] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (X),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0107] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (Xa):

##STR00025##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, ethanolamine, serine, inositol, choline, or
glycerol; Y is either nothing or a spacer group ranging in length from 2
to 30 atoms; X is a physiologically acceptable monomer, dimer, oligomer,
or polymer wherein X is a glycosaminoglycan; and n is a number from 1 to
1000 or a number from 2 to 1000; wherein any bond between the ceramide
phosphoryl, Z, Y and X is either an amide or an esteric bond.

[0108] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (Xa),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0109] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XI):

##STR00026##

wherein is R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Y is either nothing or a spacer group ranging in length from 2 to
30 atoms; X is a physiologically acceptable monomer, dimer, oligomer or
polymer wherein X is a glycosaminoglycan; and n is a number from 1 to
1000 or a number from 2 to 1000; wherein if Y is nothing the sphingosyl
is directly linked to X via an amide bond and if Y is a spacer, the
spacer is directly linked to X and to the sphingosyl via an amide bond
and to X via an amide or an esteric bond.

[0110] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XI),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0111] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XII):

##STR00027##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, ethanolamine, serine, phosphate, inositol,
choline, or glycerol; Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a
number from 1 to 1000 or a number from 2 to 001000; wherein any bond
between the ceramide, Z, Y and X is either an amide or an esteric bond.

[0112] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XII),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0113] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIIa):

##STR00028##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms: Z is either nothing, ethanolamine, serine, inositol, phosphate,
choline, or glycerol; Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a
number from 1 to 1000 or a number from 2 to 001000; wherein any bond
between the ceramide, Z, Y and X is either an amide or an esteric bond.

[0114] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIIa),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0115] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIII):

##STR00029##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, choline, ethanolamine, serine, phosphate,
inositol, or glycerol; Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a
number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the diglyceryl, Z, Y and X is either an amide or an esteric bond.

[0116] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIII),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0117] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIV):

##STR00030##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, choline, ethanolamine, serine, phosphate,
inositol, or glycerol; Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a
number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the glycerolipid, Z, Y and X is either an amide or an esteric
bond.

[0118] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIV),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0119] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XV):

##STR00031##

wherein R1 is a linear, saturated, mono-unsaturated, or
poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, choline, ethanolamine, serine,
phosphate, inositol, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
n is a number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the glycerolipid, Z, Y and X is either an amide or an esteric
bond.

[0120] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XV),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0121] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XVI):

##STR00032##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms: R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, choline, ethanolamine, serine, phosphate,
inositol, or glycerol; Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a
number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the lipid, Z, Y and X is either an amide or an esteric bond.

[0122] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XVI),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0123] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XVII):

##STR00033##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is a linear, saturated, mono-unsaturated,
or poly-unsaturated, alkyl chain ranging in length from 2 to 30 carbon
atoms; Z is either nothing, choline, ethanolamine, serine, phosphate,
inositol, or glycerol; Y is either nothing or a spacer group ranging in
length from 2 to 30 atoms; X is a physiologically acceptable monomer,
dimer, oligomer or polymer wherein X is a glycosaminoglycan; and n is a
number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the lipid, Z, Y and X is either an amide or an esteric bond.

[0124] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XVII),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0125] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XVIII):

##STR00034##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms: Z is either nothing, choline, ethanolamine, serine,
phosphate, inositol, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
n is a number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the lipid, Z, Y and X is either an amide or an esteric bond.

[0126] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XVIII),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0127] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIX):

##STR00035##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms: R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, choline, ethanolamine, seine,
phosphate, inositol, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
n is a number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the lipid, Z, Y and X is either an amide or an esteric bond.

[0128] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XIX),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0129] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XX):

##STR00036##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, choline, ethanoleamine,
serine, phosphate, inositol, or glycerol; Y is either nothing or a spacer
group ranging in length from 2 to 30 atoms; X is a physiologically
acceptable monomer, dimer, oligomer or polymer wherein X is a
glycosaminoglycan; and n is a number from 1 to 1000 or a number from 2 to
1000; wherein any bond between the lipid, Z, Y and X is either an amide
or an esteric bond.

[0130] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XX),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0131] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XXI):

##STR00037##

wherein R1 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; R2 is either hydrogen or a linear, saturated,
mono-unsaturated, or poly-unsaturated, alkyl chain ranging in length from
2 to 30 carbon atoms; Z is either nothing, choline, ethanolamine, serine,
phosphate, inositol, or glycerol; Y is either nothing or a spacer group
ranging in length from 2 to 30 atoms; X is a physiologically acceptable
monomer, dimer, oligomer or polymer wherein X is a glycosaminoglycan; and
n is a number from 1 to 1000 or a number from 2 to 1000; wherein any bond
between the lipid, Z, Y and X is either an amide or an esteric bond.

[0132] In another embodiment, the compound for use in the present
invention is represented by the structure of the general formula (XXI),
wherein X is glycosaminoglycan (GAG) and n is a number from 1 to 70. In
another embodiment, the molecular weight of said GAG is between 5 to 20
kD.

[0133] For any or all of the compounds represented by the structures of
the general formulae (A), (I), (II), (III), (IV), (V), (VI), (VII),
(VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa), (XIII),
(XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), and (XXII)
hereinabove: In one embodiment, X is a glycosaminoglycan. According to
this aspect and in one embodiment, the glycosaminoglycan may be, inter
alia, hyaluronic acid, heparin, heparan sulfate, chondroitin sulfate,
keratin, keratan sulfate, dermatan sulfate or a derivative thereof. In
one embodiment, the chondroitin sulfate may be, inter alia,
chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. In
another embodiment, X is not a glycosaminoglycan. In another embodiment,
X is a polysaccharide, which in one embodiment is a
hetero-polysaccharide, and in another embodiment, is a
homo-polysaccharide. In another embodiment, X is a polypyranose.

[0134] In another embodiment, the glycosaminoglycan is a polymer of
disaccharide units. In another embodiment, the number of the disaccharide
units in the polymer is m. In another embodiment, m is a number from
2-10,000. In another embodiment, m is a number from 2-500. In another
embodiment, m is a number from 2-1000. In another embodiment, m is a
number from 50-500. In another embodiment, m is a number from 2-2000. In
another embodiment, m is a number from 500-2000. In another embodiment, m
is a number from 1000-2000. In another embodiment, m is a number from
2000-5000. In another embodiment, m is a number from 3000-7000. In
another embodiment, m is a number from 5000-10,000. In another
embodiment, a disaccharide unit of a glycosaminoglycan may be bound to
one lipid or phospholipid moiety. In another embodiment, each
disaccharide unit of the glycosaminoglycan may be bound to zero or one
lipid or phospholipid moieties. In another embodiment, the lipid or
phospholipid moieties are bound to the --COOH group of the disaccharide
unit. In another embodiment, the bond between the lipid or phospholipid
moiety and the disaccharide unit is an amide bond.

[0135] In one embodiment of the invention, V is nothing. Non-limiting
examples of suitable divalent groups forming the optional bridging group
(which in one embodiment, is referred to as a spacer) Y, according to
embodiments of the invention, are straight or branched chain alkylene,
e.g., of 2 or more, preferably 4 to 30 carbon atoms, --CO-alkylene-CO,
--NH-alkylene-NH--, --CO-alkylene-NH--, --NH-alkylene-NH,
CO-alkylene-NH--, an amino acid, cycloalkylene, wherein alkylene in each
instance, is straight or branched chain and contains 2 or more,
preferably 2 to 30 atoms in the chain,
--(--O--CH(CH3)CH2--)x-- wherein x is an integer of 1 or
more.

[0136] In one embodiment of the invention, the sugar rings of the
glycosaminoglycan are intact. In another embodiment, intact refers to
closed. In another embodiment, intact refers to natural. In another
embodiment, intact refers to unbroken.

[0137] In one embodiment of the invention, the structure of the lipid or
phospholipid in any compound according to the invention is intact. In
another embodiment, the natural structure of the lipid or phospholipids
in any compound according to the invention is maintained.

[0138] In some embodiments, the compounds (A), (III), (IV), (V), (VI),
(VII), (VIII), (IX), (IXa), (IXb), (X), (Xa), (XI), (XII), (XIIa),
(XIII), (XIV), (XV), (XVI), (XVII), (XVIII), (XIX), (XX) and (XXI) as
presented hereinabove comprises a Z group. In one embodiment, Z is a
nothing. In another embodiment Z is inositol. In another embodiment, Z is
choline. In another embodiment, Z is glycerol.

[0139] In some embodiments, the compounds (XII), (XIIa), (XIII), (XIV),
(XV), (XVI), (XVII), (XVIII), (XIX), (XX) and (XXI) as presented
hereinabove comprises a Z group. In one embodiment, the Z is a phosphate.
In another embodiment, the phosphate is phoso-ethanolamine
--P(OH)(═O)--O--CH2CH2--NH--. In another embodiment, the
phosphate is phospho-serine-P(OH)(═O)--O--CH2CH(COOH)--NH--.

[0141] The polymers used as starting material to prepare the lipids or
PL-conjugates may vary in molecular weight from 1 to 2,000 kDa.

[0142] In another embodiment, the phospholipid (PL)-conjugate compound of
this invention is a phosphatidylethanolamine, a phosphatidylserine, a
phosphatidylcholine, a phosphatidylinositol, a phosphatidic acid or a
phosphatidylglycerol. In another embodiment, PL comprises the residue of
palmitic acid, myristic acid, myristoleic acid, palmitoleic acid, oleic
acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic
acid, erucic acid or docosahexaenoic acid. In another embodiment, PL is
dimyristoyl phosphatidylethanolamine. In another embodiment, PL is
dipalmitoyl phosphatidylethanolamine. Phosphatidylserine (PS) and its
analogues, such as palmitoyl-stearoyl-PS, natural PS from various
sources, semisynthetic PSs, synthetic, natural and artificial PSs and
their isomers.

[0143] In one embodiment, the compounds of this invention comprise lipid
conjugates. In another embodiment, the lipid is lysophospholipids,
sphingomyelins, lysosphingomyelins, ceramide, and sphingosine.

[0144] For PE-conjugates and PS-conjugates, the phospholipid is linked to
the conjugated monomer or polymer moiety through the nitrogen atom of the
phospholipid polar head group, either directly or via a spacer group. For
PC, PI, and PG conjugates, the phospholipid is linked to the conjugated
monomer or polymer moiety through either the nitrogen or one of the
oxygen atoms of the polar head group, either directly or via a spacer
group. The PS can bind also via the COOH group.

[0145] In one embodiment, the lipid and PL are conjugated to
glycosaminoglycan (GAG). In another embodiment, the GAG is hyaluronic
acid, heparin, heparan sulfate, chondroitin, chondroitin sulfate,
dermatan sulfate or keratan sulfate. In another embodiment, GAG is
hyaluronic acid. In another embodiment, GAG is heparin. In another
embodiment, GAG is chondroitin. In another embodiment, GAG is chondroitin
sulfate. In another embodiment, GAG is dermatan sulfate, in another
embodiment, GAG is keratan sulfate.

[0146] In another embodiment, chondroitin sulfate is
chondroitin-6-sulfate, chondroitin-4-sulfate or a derivative thereof. In
another embodiment, dermatan sulfate is dermatan-6-sulfate,
dermatan-4-sulfate or a derivative thereof.

[0147] In one embodiment, the compounds for use in the present invention
are biodegradable.

[0148] In one embodiment, the compound according to the invention is
phosphatidylethanolamine bound to aspirin. In one embodiment, the
compound according to the invention is phosphatidylethanolamine bound to
glutarate.

[0149] In some embodiments, the compounds for use are as listed in Table 1
below.

[0150] In one embodiment of the invention, the compounds for use in the
present invention are any one or more of Compounds I-LXXXVIII. In another
embodiment, the compounds for use in the present invention are Compound
XXII, Compound XXIII, Compound XXIV, Compound XXV, Compound XXVI,
Compound XXVII, Compound XXVIII, Compound XXIX, Compound XXX, or
pharmaceutically acceptable salts thereof, in combination with a
physiologically acceptable carrier or solvent. According to embodiments
of the invention, these polymers, when chosen as the conjugated moiety,
may vary in molecular weights from 200 to 2,000,000 Daltons. In one
embodiment of the invention, the molecular weight of the polymer as
referred to herein is from 200 to 1000 Daltons. In another embodiment,
the molecular weight of the polymer as referred to herein is from 200 to
1000 Daltons. In another embodiment, the molecular weight of the polymer
as referred to herein is from 1000 to 5000 Daltons. In another
embodiment, the molecular weight of the polymer as referred to herein is
from 5000 to 10,000 Daltons. In another embodiment, the molecular weight
of the polymer as referred to herein is from 10,000 to 20,000 Daltons. In
another embodiment, the molecular weight of the polymer as referred to
herein is from 10,000 to 50,000 Daltons. In another embodiment, the
molecular weight of the polymer as referred to herein is from 20,000 to
70,000 Daltons. In another embodiment, the molecular weight of the
polymer as referred to herein is from 50,000 to 100,000 Daltons. In
another embodiment, the molecular weight of the polymer as referred to
herein is from 100,000 to 200,000 Daltons. In another embodiment, the
molecular weight of the polymer as referred to herein is from 200,000 to
500,000 Daltons. In another embodiment, the molecular weight of the
polymer as referred to herein is from 200,000 to 1,000,000 Daltons. In
another embodiment, the molecular weight of the polymer as referred to
herein is from 500,000 to 1,000,000 Daltons. In another embodiment, the
molecular weight of the polymer as referred to herein is from 1,000,000
to 2,000,000 Daltons. Various molecular weight species have been shown to
have the desired biological efficacy.

[0151] Examples of suitable divalent groups forming the optional bridging
group Y are straight- or branched-chain alkylene, e.g., of 2 or more,
preferably 4 to 18 carbon atoms, --CO-alkylene-CO, --NH-alkylene-NH--,
--CO-alkylene-NH--, cycloalkylene, wherein alkylene in each instance, is
straight or branched chain and contains 2 or more, preferably 2 to 18
carbon atoms in the chain, --(--O--CH(CH3)CH2--)x--
wherein x is an integer of 1 or more.

[0152] In another embodiment, in addition to the traditional phospholipid
structure, related derivatives for use in this invention are
phospholipids modified at the C1 or C2 position to contain an ether or
alkyl bond instead of an ester bond. In one embodiment of the invention,
the alkyl phospholipid derivatives and ether phospholipid derivatives are
exemplified herein. In one embodiment, these derivatives are exemplified
hereinabove by the general formulae (VIII) and (IX).

[0153] In one embodiment of the invention, X is covalently conjugated to a
lipid. In another embodiment, X is covalently conjugated to a lipid via
an amide bond. In another embodiment, X is covalently conjugated to a
lipid via an esteric bond. In another embodiment, the lipid is
phosphatidylethanolamine.

[0154] In one embodiment, cell surface GAGs play a key role in protecting
cells from diverse damaging agents and processes, such as reactive oxygen
species and free radicals, endotoxins, cytokines, invasion promoting
enzymes, and agents that induce and/or facilitate degradation of
extracellular matrix and basal membrane, cell invasiveness, white cell
extravasation and infiltration, chemotaxis, and others. In addition, cell
surface GAGs protect cells from bacterial, viral and parasitic infection,
and their stripping exposes the cell to interaction and subsequent
internalization of the microorganism. Enrichment of cell surface GAGs
would thus assist in protection of the cell from injurious processes.
Thus, in one embodiment of the invention, PLA2 inhibitors are conjugated
to GAGs or GAG-mimicking molecules. In another embodiment, these
Lipid-conjugates provide wide-range protection from diverse injurious
processes, and ameliorate diseases that require cell protection from
injurious biochemical mediators.

[0155] In another embodiment, a GAG-mimicking molecule may be, inter alia,
a negatively charged molecule. In another embodiment, a GAG-mimicking
molecule may be, inter alia, a salicylate derivative. In another
embodiment, a GAG-mimicking molecule may be, inter alia, a dicarboxylic
acid.

[0157] In another embodiment, the antibacterial agent is selected from a
wide range of therapeutic agents and mixtures of therapeutic agents which
may be administered in sustained release or prolonged action form.
Nonlimiting illustrative specific examples of antibacterial agents
include bismuth containing compounds, sulfonamides; nitrofurans,
metronidazole, tinidazole, nimorazole, benzoic acid; aminoglycosides,
macrolides, penicillins, polypeptides, tetracyclines, cephalosporins,
chloramphenicol, and clindamycin. Preferably, the antibacterial agent is
selected from the group consisting of bismuth containing compounds, such
as, without limitation, bismuth aluminate, bismuth subcitrate, bismuth
subgalate, bismuth subsalicylate, and mixtures thereof; the sulfonamides;
the nitrofurans, such as nitrofurazone, nitrofurantoin, and furozolidone;
and miscellaneous antibacterials such as metrotidazole, tinidazole,
nimorazole, and benzoic acid; and antibiotics, including the
aminoglycosides, such as gentamycin, neomycin, kanamycin, and
streptomycin; the macrolides, such as erythromycin, clindamycin, and
rifamycin; the penicillins, such as penicillin G, penicillin V,
Ampicillin and amoxicillin; the polypeptides, such as bactracin and
polymyxin; the tetracyclines, such as chlorotetracycline,
oxytetracycline, and doxycycline; the cephalospoins, suck as cephalexin
and cephalothin; and miscellaneous antibiotics, such as chloramphenicol,
and clindamycin. More preferably, the antibacterial agent is selected
from the group consisting of bismuth aluminate, nitrofurantoin,
furozolidone, metronidazole, tinidazole, nimorazole, benzoic acid,
gentamycin, neomycin, kanamycin, streptomycin, erythromycin, clindamycin,
rifamycin, penicillin G, penicillin V, Ampicillin amoxicillin,
bacitracin, polymyxin, tetracycline, chlorotetracycline, oxytetracycline,
doxycycline, cephalexin, cephalothin, chloramphenicol, and clidamycin.

[0158] In another embodiment, the antifungal agent is astemizole,
clotrimazole, omeprazole, econazole, oxiconazole, sulconazole,
fluconazole, ketoconazole, itraconazole, torbinafine, and mixtures
thereof. In another embodiment, a composition as described herein
comprises a calcium channel blocker.

[0159] In another embodiment, the invention provides a pharmaceutical
composition comprising a lipid or phospholipid moiety bonded to a
physiologically acceptable monomer, dimer, oligomer, or polymer; and a
pharmaceutically acceptable carrier or excipient. In another embodiment,
the invention provides a pharmaceutical composition comprising a
conjugate as described for treating a subject afflicted with a tumor. In
another embodiment, the invention provides a pharmaceutical composition
comprising a conjugate as described for treating a subject in risk of
developing a tumor. In another embodiment, the invention provides a
pharmaceutical composition comprising a conjugate as described for
inhibiting MMP production and/or MMP activity in a cell. In another
embodiment, the invention provides a pharmaceutical composition
comprising a conjugate as described for treating a subject afflicted with
atherosclerosis. In another embodiment, a pharmaceutical composition
comprising a conjugate as described is effective in inhibiting blood
vessels formation. In another embodiment, a pharmaceutical composition
comprising a conjugate as described is effective in inhibiting
endothelial cell migration. In another embodiment, a pharmaceutical
composition comprising a conjugate as described counteracts the effect of
MMP.

[0160] In another embodiment, the invention provides a pharmaceutical
composition comprising a combination of active pharmaceutical ingredients
comprising a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer; and an anti-cancer
agent. In another embodiment, the invention provides a pharmaceutical
composition comprising a combination of active pharmaceutical ingredients
comprising a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer; and a an anti-tumor
agent. In another embodiment, the invention provides a pharmaceutical
composition comprising a combination of active pharmaceutical ingredients
comprising a lipid or phospholipid moiety bonded to a physiologically
acceptable monomer, dimer, oligomer, or polymer; and a cardiovascular
therapeutic agent.

[0161] In another embodiment, the invention provides a pharmaceutical
composition for treating a subject afflicted with cancer characterized by
tumors or afflicted with atherosclerosis, including any one of the
compounds for use in the present invention or any combination thereof;
and a pharmaceutically acceptable carrier or excipient. In another
embodiment, the compounds for use in the present invention include, inter
alia, the compounds represented by the structures of the general formulae
as described hereinbelow: (A), (I), (II), (III), (IV), (V), (VI), (VII),
(VIII), (IX), (IXa), (IXb), (X), (Xa) (XI), (XII), (XIIa), (XIII), (XIV),
(XV), (XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or any
combination thereof.

Preparation of Compounds for Use in the Present Invention

[0162] In one embodiment, the preparation of high molecular weight
Lipid-conjugates for use in the methods of the present invention is as
described in U.S. Pat. No. 5,064,817, which is incorporated fully herein
by reference. In one embodiment, these synthetic methods are applicable
to the preparation of Lipid-conjugates as well, i.e. Lipid-conjugates
comprising monomers and dimers as the conjugated moiety, with appropriate
modifications in the procedure as would be readily evident to one skilled
in the art. The preparation of some Lipid-conjugates may be conducted
using methods well known in the art or as described in U.S. Provisional
Patent Application 60/704,874, which is incorporated herein by reference
in its entirety.

Dosages and Routes of Administration

[0163] The methods of this invention can be adapted to the use of the
therapeutic compositions comprising Lipid-conjugates or
Phospholipid-conjugates in admixture with conventional excipients, i.e.
pharmaceutically acceptable organic or inorganic carrier substances
suitable for parenteral, enteral (e.g., oral) or topical application
which do not deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include but are not limited to
water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl
alcohols, polyethylene glycols, gelatine, carbohydrates such as lactose,
amylose or starch, magnesium stearate, talc, silicic acid, viscous
paraffin, white paraffin, glycerol, alginates, hyaluronic acid, collagen,
perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol
fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc.
The pharmaceutical preparations can be sterilized and if desired mixed
with auxiliary agents, e.g., lubricants, preservatives, stabilizers,
wetting agents, emulsifiers, salts for influencing osmotic pressure,
buffers, coloring, flavoring and/or aromatic substances and the like
which do not deleteriously react with the active compounds. They can also
be combined where desired with other active agents, e.g., vitamins,
steroids, anti-inflammatory compounds, etc., as will be understood by one
skilled in the art.

[0164] In one embodiment, the route of administration may be parenteral,
enteral, or a combination thereof. In another embodiment, the route may
be intra-ocular, conjunctival, topical, transdermal, intradermal,
subcutaneous, intraperitoneal, intravenous, intra-arterial, vaginal,
rectal, intratumoral, parcanceral, transmucosal, intramuscular,
intravascular, intraventricular, intracranial, inhalation, nasal
aspiration (spray), sublingual, oral, aerosol or suppository or a
combination thereof. In one embodiment, the dosage regimen will be
determined by skilled clinicians, based on factors such as exact nature
of the condition being treated, the severity of the condition, the age
and general physical condition of the patient, etc.

[0165] In another embodiment, the compositions include those suitable for
oral, rectal, intravaginal, topical, nasal, ophthalmic or parenteral
administration, all of which may be used as routes of administration
using the materials of the present invention. Other suitable routes of
administration include direct injection onto an arterial surface and
intraparenchymal injection directly into targeted areas of an organ or a
tumor. The term "parenteral" includes subcutaneous injections,
intravenous, intramuscular, intrasternal injection or infusion
techniques.

[0166] The compositions may conveniently be presented in unit dosage form
and may be prepared by any of the methods well known in the art of
pharmacy. Methods typically include the step of bringing the active
ingredients of the invention into association with a carrier which
constitutes one or more accessory ingredients.

[0167] Compositions of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets, tablets or lozenges, each containing a predetermined amount of
the compounds of the invention in liposomes or as a suspension in an
aqueous liquid or non-aqueous liquid such as a syrup, an elixir, or an
emulsion.

[0168] Compositions suitable for parenteral administration conveniently
comprise a sterile aqueous preparation of the molecule of the invention
which is preferably isotonic with the blood of the recipient. This
aqueous preparation may be formulated according to known methods using
those suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation may also be a sterile injectable solution
or suspension in a non-toxic parenterally-acceptable diluent or solvent,
for example as a solution in 1,3-butane diol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's solution
and isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For this
purpose any bland fixed oil may be employed including synthetic mono- or
diglycerides. In addition, fatty acids such as oleic acid find use in the
preparation of injectables.

[0169] Oral agents provide the advantages of easy administration and
chronic systemic treatment. However, local delivery of MMP inhibitors via
catheters, gene transfer techniques, and endovascular stents or polymers
can be utilized in order to control localized disease.

[0170] An exemplary pharmaceutical composition is a therapeutically
effective amount of a composition as described herein will inhibit MMP as
shown in a standard assay, which optionally is included in a
pharmaceutically-acceptable and compatible carrier.

[0171] The term "pharmaceutically-acceptable and compatible carrier" as
used herein, includes one or more compatible solid or liquid filler
diluents or encapsulating substances that are suitable for administration
to a human or other animal. In the present invention, the term "carrier"
thus denotes an organic or inorganic ingredient, natural or synthetic,
with which the compounds of the invention are combined to facilitate
application. The term "therapeutically-effective amount" is that amount
of the present pharmaceutical composition which produces a desired result
or exerts a desired influence on the particular condition being treated.
In another embodiment, when the composition is being used as prophylactic
additional doses will be administered at periodic intervals after the
initial administration. Various concentrations may be used in preparing
compositions incorporating the same ingredient to provide for variations
in the age of the patient to be treated, the severity of the condition,
the duration of the treatment and the mode of administration.

[0172] The term "compatible", as used herein, means that the components of
the pharmaceutical compositions are capable of being commingled with a
small molecule of the present invention, and with each other, in a manner
such that does not substantially impair the desired pharmaceutical
efficacy.

[0173] Doses of the pharmaceutical compositions of the invention will vary
depending on the subject and upon the particular route of administration
used. Dosages can range from 0.1 to 100,000 μg/kg per day, more
preferably 1 to 10,000 μg/kg. By way of an example only, an overall
dose range of from about, for example, 1 microgram to about 300
micrograms might be used for human use. This dose can be delivered at
periodic intervals based upon the composition. In another embodiment,
compounds might be administered daily. Pharmaceutical compositions of the
present invention can also be administered to a subject according to a
variety of other, well-characterized protocols. For example, using pulsed
therapy.

[0174] In general, the doses utilized for the above described purposes
will vary, but will be in an effective amount to exert the desired
anti-disease effect. As used herein, the term "pharmaceutically effective
amount" refers to an amount of a compound of formulae I-XXI which will
produce the desired alleviation in symptoms or signs of disease in a
patient. The doses utilized for any of the above-described purposes will
generally be from 1 to about 1000 milligrams per kilogram of body weight
(mg/kg), administered one to four times per day, or by continuous IV
infusion. When the compositions are dosed topically, they will generally
be in a concentration range of from 0.1 to about 10% w/v, administered
1-4 times per day.

[0175] Desired time intervals for delivery of multiple doses of a
particular composition can be determined by one of ordinary skill in the
art employing no more than routine experimentation. The conjugate can be
comprised of non-antigenic polymeric substances such as dextran,
polyvinyl pyrrolidones, polysaccharides, starches, polyvinyl alcohols,
polyacryl amides or other similar substantially non-immunogenic polymers.
Polyethylene glycol (PEG) is preferred. Other poly(alkylenes oxides)
include monomethoxy-polyethylene glycol polypropylene glycol, block
copolymers of polyethylene glycol, and polypropylene glycol and the like.
The polymers can also be distally capped with C1-4 alkyls instead of
monomethoxy groups. The poly(alkylene oxides) used must be soluble in
liquid at room temperature. Thus, they preferably have a molecular weight
from about 200 to about 20,000 daltons, more preferably about 2,000 to
about 10,000 and still more preferably about 5,000.

[0177] In one embodiment, the use of a single chemical entity with potent
anti-oxidant, membrane-stabilizing, anti-proliferative, anti-chemokine,
anti-migratory, and anti-inflammatory activity provides the desired
protection for a subject afflicted with arthritis, or in another
embodiment, the methods of this invention provide for use of a
combination of the compounds described. In another embodiment, the
compounds for use in the present invention may be provided in a single
formulation/composition, or in another embodiment, multiple formulations
may be used. In one embodiment, the formulations for use in the present
invention may be administered simultaneously, or in another embodiment,
at different time intervals, which may vary between minutes, hours, days,
weeks or months.

[0178] In one embodiment the compositions comprising the compounds for use
in the present invention may be administered via different routes, which
in one embodiment, may be tailored to provide different compounds at
different sites, for example some compounds may be given by inta-joint
injection to provide for superior relief in-situ, and in another
embodiment, some formulations/compounds/compositions may be provided via
various topical formulations, or in another embodiment, systemically, to
provide for broader effect.

[0179] In one embodiment, the compounds for use in the invention may be
used for acute treatment of temporary conditions, or may be administered
chronically, as needed. In one embodiment of the invention, the
concentrations of the compounds will depend on various factors, including
the nature of the condition to be treated, the condition of the patient,
the route of administration and the individual tolerability of the
compositions.

[0180] In one embodiment, the methods of this invention provide for the
administration of the compounds throughout the life of the subject, or in
another embodiment, episodically, in response to severity or constancy of
symptomatic stages, or in another embodiment, at the onset of pain
associated with arthritis. In another embodiment, the patients to whom
the lipid or PL conjugates should be administered are those that are
experiencing symptoms of disease or who are at risk of contracting the
disease or experiencing a recurrent episode or exacerbation of the
disease, or pathological conditions associated with the same.

[0181] As used herein, the term "pharmaceutically acceptable carrier"
refers to any formulation which is safe, and provides the appropriate
delivery for the desired route of administration of an effective amount
of at least one compound of the present invention. As such, all of the
above-described formulations of the present invention are hereby referred
to as "pharmaceutically acceptable carriers." This term refers to as well
the use of buffered formulations wherein the pH is maintained at a
particular desired value, ranging from pH 4.0 to pH 9.0, in accordance
with the stability of the compounds and route of administration.

[0182] For parenteral application, particularly suitable are injectable,
sterile solutions, preferably oily or aqueous solutions, as well as
suspensions, emulsions, or implants, including suppositories. Ampoules
are convenient unit dosages.

[0183] For topical application, particularly for the treatment of skin
diseases such as but not limited to contact dermatitis or psoriasis,
admixture of the compounds with conventional creams or delayed release
patches is acceptable.

[0184] For enteral application, particularly suitable are tablets,
dragees, liquids, drops, suppositories, or capsules. Syrup, elixir, or
the like can be used when a sweetened vehicle is employed. When
indicated, suppositories or enema formulations may be the recommended
route of administration.

[0185] Sustained or directed release compositions can be formulated, e.g.,
liposomes or those wherein the active compound is protected with
differentially degradable coatings, e.g., by microencapsulation, multiple
coatings, etc. It is also possible to freeze-dry the new compounds and
use the lyophilisates obtained, for example, for the preparation of
products for injection.

[0186] Articular injection are used for treating an osteoarthritic joint
with at least one of the compounds as described herein in a concentration
of 1-50 mg/ml in a volume of 1-10 ml/injection.

[0187] The compounds as described herein may be administered in different
ways, for example, periarticular injection, peritendonous injection,
periligamentous injection or intramuscular perfusion. Methods of making
such injections are known to one of ordinary skill in the art. Such
injections are generally subcutaneous and target the vicinity of a joint,
especially near the insertions or origins of muscle tendons and
ligaments. Local analgesics may be provided at the site of injection.
Such analgesics are known to one of ordinary skill in the art.

[0188] Further active substances that can be used in an injectable dosage
form are: anti-cancer drugs, small molecules, antibiotics, antiseptics,
sodium hyaluronate, a glucocorticoidor any combination thereof.
Excipients include but are not limited to: isotonizing agents, such as
sodium chloride, mannitol, or sorbitol, water for injection as solvent,
sodium monohydrogenphosphate, and sodium dihydrogenphosphate. The
solution may additionally contain pH modifiers, such as sodium hydroxide,
sodium hydrogenphosphate, hydrochloric acid, or citric acid, surfactants,
such as polysorbate 80; sodium edetate as stabilizer (synergistic
anti-oxidative agent); propylene glycol or polyethylene glycol as
cosolvent; and/or antimicrobial agents, like benzyl alcohol, methyl- and
propyl-4-hydroxybenzoate, or cetylpyridinium chloride. In the treatment
of larger joints, such as the knee, hip or shoulder, syringes of 10-40
mg/2.0 ml are used.

[0189] Suspension formulations additionally contain stabilizers, such as
carmellose sodium, hypromellose or gelatine, to avoid or reduce the
sedimentation of the suspension as far as possible, and to allow for a
fast and reliable re-dispersion of the suspension prior to application.
It is essential that the crystals in the suspension formulations maintain
their particle size. An uncontrolled growth of crystals bears the risk of
poor biocompatibility of the suspension formulation upon intra-articular
injection.

[0190] The injectable formulations can be also formulated as a dry powder
which has to be re-dispersed by addition of the dispersing medium (e.g.,
water for injection). For suspension formulations, it is essential that
they are re-dispersed directly before the application, and that the
resulting suspension appears homogenous.

[0191] It will be appreciated that the actual preferred amounts of active
compound in a specific case will vary according to the specific compound
being utilized, the particular compositions formulated, the mode of
application, and the particular situs and organism being treated. Dosages
for a given host can be determined using conventional considerations,
e.g., by customary comparison of the differential activities of the
subject compounds and of a known agent, e.g., by means of an appropriate,
conventional pharmacological protocol.

[0192] In another embodiment, provided herein a method for inhibiting a
MMP production in a cell, comprising contacting the cell with a
composition comprising a compound of the invention. In another
embodiment, provided herein a method for inhibiting MMP 2 production in a
cell, comprising contacting the cell with a composition comprising a
compound of the invention. In another embodiment, provided herein a
method for inhibiting MMP 9 production in a cell, comprising contacting
the cell with a composition comprising a compound of the invention. In
another embodiment, provided herein a method for inhibiting a MMP
production in a malignant cell, comprising contacting the cell with a
composition comprising a compound of the invention. In another
embodiment, provided herein a method for inhibiting a MMP production in a
cell expressing elevated level of a MMP, comprising contacting the cell
with a composition comprising a compound of the invention.

[0193] In another embodiment, provided herein a method for inhibiting
invasiveness of a cancer cell, comprising the step of contacting a cancer
cell with a composition comprising a compound of the invention. In
another embodiment, provided herein a method for inhibiting the
production of a MMP in a cell, comprising the step of contacting the
cancer cell with a composition comprising a compound as described herein.
In another embodiment, provided herein a method for inhibiting the
production of a MMP in a cancer cell, comprising the step of contacting
the cancer cell with a composition comprising a compound as described
herein. In another embodiment, provided herein a method for inhibiting
the production of a MMP in a cell, comprising the step of contacting the
cell with a composition comprising a compound as described herein. In
another embodiment, provided herein a method for inhibiting the
expression of a MMP in a cell, comprising the step of contacting the cell
with a composition comprising a compound as described herein. In another
embodiment, provided herein a method for inhibiting the transcription of
a MMP in a cell, comprising the step of contacting the cell with a
composition comprising a compound as described herein. In another
embodiment, provided herein a method for inhibiting the activation of a
pre-MMP in a cell, comprising the step of contacting the cell with a
composition comprising a compound as described herein.

[0194] In another embodiment, provided herein a method for inhibiting a
collagenolytic activity of a MMP, comprising the step of contacting a MMP
with a composition comprising a compound as described herein. In another
embodiment, provided herein a method for inhibiting a collagenolytic
activity of a MMP, comprising the step of contacting a metastatic cell
with a composition comprising a compound as described herein. In another
embodiment, provided herein a method for inhibiting a collagenolytic
activity of a MMP, comprising the step of contacting an endothelial cell
with a composition comprising a compound as described herein. In another
embodiment, provided herein a method for inhibiting a collagenolytic
activity of a cell by contacting the cell with a MMP inhibitor as
described herein. In another embodiment, provided herein a method for
inhibiting a collagenolytic activity of an endothelial cell by contacting
the cell with a MMP inhibitor as described herein. In another embodiment,
provided herein a method for inhibiting a collagenolytic activity of a
malignant cell by contacting the cell with a MMP inhibitor as described
herein.

[0195] In another embodiment, provided herein a method for treating a
subject afflicted with a disease in which increased production of a MMP
is associated with the disease, comprising the step of administering to
the subject a composition comprising a PL compound as described
hereinabove. In another embodiment, provided herein a method for treating
a subject afflicted with a disease in which increased activity of a MMP
is associated with the disease, comprising the step of administering to
the subject a composition comprising a PL compound as described
hereinabove. In another embodiment, provided herein a method for treating
a subject suffering from a medical condition in which increased
production of a MMP causes the medical condition, comprising the step of
administering to the subject a composition comprising a PL compound as
described hereinabove. In another embodiment, provided herein a method
for treating a subject suffering from a medical condition in which
increased activity of a MMP causes the medical condition, comprising the
step of administering to the subject a composition comprising a PL
compound as described hereinabove. In another embodiment, provided herein
a method for treating a subject suffering from a medical condition in
which increased production of a MMP is associated with the medical
condition, comprising the step of administering to the subject a
composition comprising a PL compound as described hereinabove. In another
embodiment, provided herein a method for treating a subject suffering
from a medical condition in which increased activity of a MMP is
associated with the medical condition, comprising the step of
administering to the subject a composition comprising a PL compound as
described hereinabove.

[0196] In another embodiment, a medical condition or a disease treatable
by the compounds of the invention is characterized by excessive MMP
activity and/or production. In another embodiment, the medical condition
or disease is selected from: Pterygium, Kerataconus, macular
degeneration, corneal melting, occlusions in the choroid, a heart
disease, arthritis, a cerebral disease, a tissue ulceration, abnormal
wound healing, a periodontal disease, a bone disease, a cancer
characterized by tumor growth, a cancer characterized by tumor metastasis
or invasion, HIV-infection, decubitus, decubitis ulcer, restenosis,
epidermolysis bullosa, sepsis, septic shock, neoplasm, psoriasis,
neovascularization, a liver disease, or multiple sclerosis.

[0199] In another embodiment, an inhibitor as described herein inhibits a
MMP and thus acting as an immunosuppressant. In another embodiment, an
inhibitor as described herein inhibits a MMP and thereby inhibits the
activity of TNF-α and/or IFN-γ production. In another
embodiment, an inhibitor as described herein inhibits a soluble MMP.

[0200] In another embodiment, a MMP inhibitor as described herein inhibits
localized degradation of existing ECM. In another embodiment, a MMP
inhibitor as described herein inhibits cytoskeletal rearrangement. In
another embodiment, an inhibitor as described herein inhibits cell
translocation. In another embodiment, a MMP inhibitor as described herein
inhibits cleavage of collagen. In another embodiment, a MMP inhibitor as
described herein inhibits cleavage of gelatin. In another embodiment, an
inhibitor as described herein inhibits a MMP in a fibroblasts, a PNL, a
macrophage, a Keratinocyte, an EC, a T-cell, or an eosinophil. In another
embodiment, a MMP inhibitor as described herein, inhibits the production
of IL1, IL10, TNF-α, TGF, FGF, PDGF, or any combination thereof.

[0201] In another embodiment, a MMP inhibitor as described herein is
administered to a subject having hi MMP levels in the blood. In another
embodiment, a MMP inhibitor as described herein is administered to a
subject having MMP levels at above a threshold level in the blood. In
another embodiment, a MMP inhibitor as described herein is administered
to a subject having above normal MMP levels in the blood. In another
embodiment, a MMP inhibitor as described herein is administered to a
subject having hi MMP levels in the urine. In another embodiment, a MMP
inhibitor as described herein is administered to a subject having MMP
levels at above a threshold level in the urine. In another embodiment, a
MMP inhibitor as described herein is administered to a subject having
above normal MMP levels in the urine.

[0202] In another embodiment, a MMP is a Zn2+ endopeptidase. In
another embodiment, a MMP is a 92 kDa gelatinase, collagenase,
stromelysin or a membrane-bound MMP. In another embodiment, a MMP is
expressed in an inflammatory condition. In another embodiment, a MMP is
capable of degrading a connective tissue. In another embodiment, a MMP is
a gelatinase such as MMP-2 and MMP-9. In another embodiment, a MMP is a
stromelysin such as MMP-3. In another embodiment, a MMP is a collagenase
such as MMP-1, MMP-8, and MMP-13 which are involved in tissue matrix
degradation and have been implicated in many pathological conditions
involving abnormal connective tissue and basement membrane matrix
metabolism.

[0203] In another embodiment, a MMP is a proteolytic enzyme. In another
embodiment, a MMP maintains the integrity of the extracellular matrix. In
another embodiment, excessive MMP activity results in loss of structural
proteins that maintain the normal architecture of an organ. In another
embodiment, excessive MMP activity results in activation of inflammatory
cells that perpetuate organ damage. In another embodiment, a MMP
activates an acute inflammatory pathway. In another embodiment, a MMP
activates a chronic inflammatory pathway, involved in liver damage.
Second, these enzymes are especially highly expressed in a variety of
liver diseases. In another embodiment, a MMP is involved in maintaining
the structural integrity of an organ. In another embodiment, a MMP is
involved in the progression of fibrogenesis.

[0204] In another embodiment, a MMP inhibitor (PL of the invention) as
described herein is used to control excessive proteolytic degradation of
the extracellular matrix. In another embodiment, a MMP inhibitor (PL of
the invention) as described herein is used to control cell invasion.

[0205] In another embodiment, a MMP inhibitor as described herein is
selective to MMP-2 and/or MMP-9. In another embodiment, a MMP inhibitor
as described herein is not selective to a particular MMP. In another
embodiment, arachidonic acid (AA)-derived metabolites regulates MMP
expression. In another embodiment, phospholipase A2, the AA
producing enzymes, regulates MMP expression.

[0206] In another embodiment, over expression pattern of MMP 2, MMP 9, MMP
13, or a combination thereof leads to the progression of liver damage. In
another embodiment, a MMP inhibitor as described herein inhibits the
progression of liver damage caused by excessive activity of MMP 2, MMP 9,
MMP 13, or a combination thereof. In another embodiment, a MMP inhibitor
as described herein inhibits the progression of liver damage caused by
excessive activity of MMP 2, MMP 9, MMP 13, or a combination thereof in
activated stellate cells. In another embodiment, a MMP inhibitor as
described herein inhibits the progression of liver damage caused by
excessive activity of MMP 2, MMP 9, MMP 13, or a combination thereof in
activated Kupffer cells. In another embodiment, a MMP inhibitor as
described herein ameliorates symptoms associated with a liver disease. In
another embodiment, a MMP inhibitor as described herein ameliorates
symptoms associated with liver damage. In another embodiment, a MMP
inhibitor as described herein is used in combination another compound or
compounds which induce mechanisms of hepatoprotection. In another
embodiment, a MMP inhibitor as described herein is used in combination
another compound or compounds which induce mechanisms of
hepatogeneration.

[0207] In another embodiment, a MMP inhibitor as described herein is
useful for the treatment of diseases related to bone or cartilage, such
as rheumatoid arthritis, osteoarthritis, etc. In another embodiment, a
MMP inhibitor as described herein is useful for inhibiting the loss of
glycoprotein and collagen in articular cartilage.

[0208] In another embodiment, a MMP inhibitor as described herein is
useful for preventing arteriosclerosis. In another embodiment, a MMP
inhibitor as described herein is useful for inhibiting the progress of
arteriosclerosis. In another embodiment, a MMP inhibitor as described
herein is useful in treating a subject afflicted with arteriosclerosis.

[0209] In another embodiment, a MMP inhibitor as described herein is
useful for preventing re-stricturization (re-stenochoria) of post
angiopoietic operation. In another embodiment, a MMP inhibitor as
described herein is useful for inhibiting the progress of
re-stricturization of post angiopoietic operation. In another embodiment,
a MMP inhibitor as described herein is useful in treating a subject
afflicted with re-stricturization (re-stenochoria) of post angiopoietic
operation.

[0210] In another embodiment, a MMP inhibitor as described herein is
useful as an etiomatic therapy. In another embodiment, a MMP inhibitor as
described herein is a MMP 13 inhibitor. In another embodiment, a MMP
inhibitor as described herein is useful for preventing bone arthritis and
rheumatoid arthritis. In another embodiment, a MMP inhibitor as described
herein is useful for inhibiting the progress of bone arthritis and
rheumatoid arthritis. In another embodiment, a MMP inhibitor as described
herein is useful in treating a subject afflicted with bone arthritis
and/or rheumatoid arthritis.

[0211] In another embodiment, a MMP inhibitor as described herein is
useful as a prophylactic and/or therapeutic treating agent.

[0212] In another embodiment, a MMP inhibitor as described herein is used
for inhibiting invasion and metastasis of malignant cells. In another
embodiment, a MMP-2 and/or MMP-9 inhibitor as described herein is used
for inhibiting invasion and metastasis of malignant cells. In another
embodiment, a MMP-2 and/or MMP-9 inhibitor as described herein is used
for inhibiting hematological malignancies. In another embodiment, a MMP
inhibitor as described herein is used for treating a subject afflicted
with acute myeloid leukemia. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with acute
myelomonocytic leukemia. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with acute
monoblastic and monocytic leukemia. In another embodiment, a MMP
inhibitor as described herein is used for treating a subject afflicted
with acute erytroid leukemia. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with acute
megakaryoblastic leukemia. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with acute
basophilic leukemia. In another embodiment, a MMP inhibitor as described
herein is used for treating a subject afflicted with acute panmyelosis
with myelofibrosis. In another embodiment, a MMP inhibitor as described
herein is used for treating a subject afflicted with myeloid sarcoma.

[0213] In another embodiment, a MMP inhibitor as described herein is used
for treating a subject afflicted with a hernia. In another embodiment, a
MMP inhibitor as described herein is used for treating a subject
afflicted with an abdominal hernia. In another embodiment, a MMP
inhibitor as described herein is used for treating a subject afflicted
with a groin hernia. In another embodiment, a MMP inhibitor as described
herein is used for reducing the risk of recurrent hernias.

[0214] In another embodiment, a MMP inhibitor as described herein is used
for treating a subject afflicted with lymphangioleiomyomatosis. In
another embodiment, a MMP inhibitor as described herein inhibits tissue
degradation in patients with lymphangioleiomyomatosis.

[0215] In another embodiment, a MMP inhibitor as described herein is used
for treating a subject suffering from pseudocyst formation. In another
embodiment, a MMP inhibitor as described herein is used for treating a
subject suffering from an accumulation of oedema. In another embodiment,
a MMP inhibitor as described herein is used for treating a subject
afflicted with sinusitis. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with chronic
sinusitis. In another embodiment, a MMP inhibitor as described herein is
used for treating a subject afflicted with nasal polyposis.

[0216] In another embodiment, a MMP inhibitor as described herein is used
for treating a subject afflicted with multiple sclerosis. In another
embodiment, a MMP inhibitor as described herein is used for ameliorating
symptoms associated with multiple sclerosis in a subject in need thereof.

[0217] In another embodiment, a MMP inhibitor as described herein is used
for treating a child afflicted with an inflammatory condition which
disrupts the elastic lamina. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with Kawasaki
disease. In another embodiment, a MMP inhibitor as described herein is
used for treating a subject afflicted with an acute type of systemic
vasculitis in children.

[0218] In another embodiment, a subject according to the invention is a
human subject. In another embodiment, a subject according to the
invention is a mammal. In another embodiment, a subject according to the
invention is a non-human mammal. In another embodiment, a subject
according to the invention is a farm animal. In another embodiment, a
subject according to the invention is a primate. In another embodiment, a
subject according to the invention is a pet. In another embodiment, a
subject according to the invention is a laboratory animal.

[0219] In another embodiment, a MMP inhibitor as described herein is used
for treating a subject afflicted with a heart disease. In another
embodiment, a MMP inhibitor as described herein is used for treating a
subject afflicted with hypertension. In another embodiment, a MMP
inhibitor as described herein is used for treating a subject afflicted
with myocardial fibrosis.

[0220] In another embodiment, a MMP inhibitor as described herein is used
for treating a subject afflicted with psoriasis. In another embodiment, a
MMP inhibitor as described herein is used for treating a subject
afflicted with cutaneous psoriasis. In another embodiment, a MMP
inhibitor as described herein is used for treating a subject afflicted
with psoriatic arthritis. In another embodiment, a MMP inhibitor as
described herein is used for treating a subject afflicted with a skin
lesion.

[0221] In another embodiment, a MMP inhibitor as described herein inhibits
the expression of a MMP. In another embodiment, a MMP inhibitor as
described herein inhibits the transcription of a MMP. In another
embodiment, a MMP inhibitor as described herein inhibits activation of a
MMP post-transcriptionally. In another embodiment, a MMP inhibitor as
described herein inhibits the activation of a MMP proenzyme. In another
embodiment, a MMP inhibitor as described herein inhibits the
collagenolytic activity of a MMP. In another embodiment, a MMP inhibitor
as described herein inhibits the collagenolytic activity of a cell. In
another embodiment, a MMP inhibitor as described herein inhibits the
collagenolytic activity of a cancer cell. In another embodiment, a MMP
inhibitor as described herein inhibits the collagenolytic activity of a
metastatic cell. In another embodiment, a MMP inhibitor as described
herein inhibits the collagenolytic activity of a tumor cell. In another
embodiment, a MMP inhibitor as described herein inhibits the metastatic
potential of a solid tumor. In another embodiment, a MMP inhibitor as
described herein inhibits a MMP in stromal cells. In another embodiment,
a MMP inhibitor as described herein inhibits a
neovascularizationiangiogenesis. In another embodiment, a MMP inhibitor
as described herein inhibits lysis of matrix surrounded by endothelial
cells thus enabling the inhibiting the invasion of new vascular
structures into a tissue. In another embodiment, a MMP inhibitor as
described herein inhibits lysis of matrix surrounded by endothelial cells
thus enabling the inhibiting the invasion of new vascular structures into
a malignant tissue.

[0222] In another embodiment, a MMP inhibitor as described herein reduces
the invasive and metastatic potential of tumor cells. In another
embodiment, a MMP inhibitor as described herein blocks the invasive
activity of cancer cells such as prostate cancer cells. In another
embodiment, a MMP inhibitor as described herein inhibits the degradation
of ECM by melanoma cells.

[0223] In another embodiment, the invention provides a method of treating
a subject afflicted with a metastatic cancer, comprising the step of
administering to the subject a composition comprising a compound of the
invention for inhibiting a MMP. In another embodiment, the invention
provides a method of treating a subject afflicted with a metastatic
cancer, comprising the step of administering to the subject a composition
comprising a compound of the invention for inhibiting MMP 2 and/or MMP 9.
In another embodiment, the invention provides a method of treating a
subject afflicted with a metastatic cancer, comprising the step of
administering to the subject a composition comprising a compound of the
invention for inhibiting a MMP in a cancerous cell. In another
embodiment, the invention provides a method of treating a subject
afflicted with a metastatic cancer, comprising the step of administering
to the subject a composition comprising a compound of the invention for
inhibiting a MMP in a malignant cell.

[0224] In another embodiment, a MMP inhibitor as described herein inhibits
MMP-9. In another embodiment, a MMP inhibitor as described herein
inhibits MMP in trophoblasts, osteoclasts, leukocytes, and their
precursors. In another embodiment, a MMP inhibitor as described herein
counteracts the activity of growth factors, cytokines, cell-cell and
cell-ECM adhesion molecules which induce MMP production and/or
activation. In another embodiment, a MMP inhibitor as described herein
inhibits a MMP metabolite. In another embodiment, a MMP inhibitor as
described herein inhibits invasion of cells through matrix barriers and
collagenolysis during invasion and tumor progression.

[0225] In some embodiments, the compounds of this invention are useful in
any application in which neoplasia or carcinogenesis is halted, modulated
or altered in any way that is beneficial to a subject in need.

[0228] In another embodiment, the subject is male. In another embodiment,
the subject is female. In some embodiments, while the methods as
described herein may be useful for treating either males or females,
females may respond more advantageously to administration of certain
compounds, for certain methods, as described and exemplified herein.

[0229] In another embodiment, the subject suffers from a sarcoma. In
another embodiment, the subject suffers from an adenocarcinoma, colon
carcinoma, melanoma, breast carcinoma, leukemia, lymphoma, gastric
carcinoma, glioblastoma, astrocytoma, bladder carcinoma, pleural
mesothelioma, oat cell carcinoma or bronchogenic carcinoma. In another
embodiment, "treating" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to prevent
or lessen the targeted pathologic condition or disorder as described
hereinabove. Thus, in another embodiment, treating may include
suppressing, inhibiting, preventing, treating, or a combination thereof.
Thus, In another embodiment, "treating" refers inter alia to increasing
time to sustained progression, expediting remission, inducing remission,
augmenting remission, speeding recovery, increasing efficacy of or
decreasing resistance to alternative therapeutics, or a combination
thereof. In another embodiment, "preventing" refers inter alia to
delaying the onset of symptoms, preventing relapse to a disease,
decreasing the number or frequency of relapse episodes, increasing
latency between symptomatic episodes, or a combination thereof. In
another embodiment, "suppressing" or "inhibiting", refers inter alia to
reducing the severity of symptoms, reducing the severity of an acute
episode, reducing the number of symptoms, reducing the incidence of
disease-related symptoms, reducing the latency of symptoms, ameliorating
symptoms, reducing secondary symptoms, reducing secondary infections,
prolonging patient survival, or a combination thereof.

[0230] In another embodiment, the terms "treating" or "treatment" includes
preventative as well as disorder remitative treatment. The terms
"reducing", "suppressing" and "inhibiting" have their commonly understood
meaning of lessening or decreasing, in another embodiment, or delaying,
in another embodiment, or reducing, in another embodiment the incidence,
severity or pathogenesis of a disease, disorder or condition. In
embodiment, the term treatment refers to delayed progression of,
prolonged remission of, reduced incidence of, or amelioration of symptoms
associated with the disease, disorder or condition. In another
embodiment, the terms "treating" "reducing", "suppressing" or
"inhibiting" refer to a reduction in morbidity, mortality, or a
combination thereof, in association with the indicated disease, disorder
or condition. In another embodiment, the term "progression" refers to an
increasing in scope or severity, advancing, growing or becoming worse.
The term "recurrence" means, in another embodiment, the return of a
disease after a remission. In another embodiment, the methods of
treatment of the invention reduce the severity of the disease, or in
another embodiment, symptoms associated with the disease, or in another
embodiment, reduces the number of biomarkers expressed during disease.

[0231] In another embodiment, the term "treating" and its included
aspects, refers to the administration to a subject with the indicated
disease, disorder or condition, or in some embodiments, to a subject
predisposed to the indicated disease, disorder or condition. The term
"predisposed to" is to be considered to refer to, inter alia, a genetic
profile or familial relationship which is associated with a trend or
statistical increase in incidence, severity, etc. of the indicated
disease. In some embodiments, the term "predisposed to" is to be
considered to refer to inter alia, a lifestyle which is associated with
increased risk of the indicated disease. In some embodiments, the term
"predisposed to" is to be considered to refer to inter alia, the presence
of biomarkers which are associated with the indicated disease, for
example, in cancer, the term "predisposed to" the cancer may comprise the
presence of precancerous precursors for the indicated cancer.

[0232] In some embodiments, the term "reducing the pathogenesis" is to be
understood to encompass reducing tissue damage, or organ damage
associated with a particular disease, disorder or condition. In another
embodiment, the term "reducing the pathogenesis" is to be understood to
encompass reducing the incidence or severity of an associated disease,
disorder or condition, with that in question. In another embodiment, the
term "reducing the pathogenesis" is to be understood to encompass
reducing the number of associated diseases, disorders or conditions with
the indicated, or symptoms associated thereto.

[0233] The term "administering", in another embodiment, refers to bringing
a subject in contact with a compound of the present invention.
Administration can be accomplished in vitro, i.e. in a test tube, or in
vivo, i.e. in cells or tissues of living organisms, for example humans.
In another embodiment, the present invention encompasses administering
the compounds of the present invention to a subject.

[0234] In another embodiment, symptoms being treated are primary, while in
another embodiment, symptoms are secondary. In another embodiment,
"primary" refers to a symptom that is a direct result of neoplasia or
carcinogenesis, while in another embodiment, "secondary" refers to a
symptom that is derived from or consequent to a primary cause. In another
embodiment, the compounds for use in the present invention treat primary
or secondary symptoms or secondary complications related to neoplasia or
carcinogenesis. In another embodiment, the compounds for use in the
present invention treat primary or secondary symptoms or secondary
complications related to neoplasia or carcinogenesis.

[0236] Thus, in one embodiment of the present invention, the compounds for
use in the present invention are directed towards the resolution of
symptoms of a disease or disorder of neoplasia or carcinogenesis. In
another embodiment, the compounds affect the pathogenesis underlying
neoplasia or carcinogenesis.

[0237] In another embodiment, neoplasia or carcinogenesis may affect a
cell, in another embodiment, a vertebrate cell, in another embodiment, a
mammalian cell, and in another embodiment, a human cell. It is to be
understood that compounds of the present invention may be efficacious in
treating any cell type in which neoplasia or carcinogenesis is present or
in which the causes of neoplasia or carcinogenesis may exert an effect.
In another embodiment, a compound for use in the present invention may
localize to or act on a specific cell type. In another embodiment, a
compound for use in the present invention may be cytoprotective. In one
embodiment a compound for use in the present invention may be inserted or
partially inserted into a cell membrane. In another embodiment a compound
for use in the present invention may be effective in treating a plurality
of cell types.

[0238] In one embodiment of the present invention, the useful
pharmacological properties of the compounds for use in the present
invention, some of which are described hereinabove, may be applied for
clinical use, and disclosed herein as methods for the prevention or
treatment of a disease. The biological basis of these methods may be
readily demonstrated by standard cellular and animal models of disease.

[0239] In another embodiment, the pharmacological activities of compounds
for use in the present invention, including membrane stabilization,
anti-inflammation, anti-oxidant action, and attenuation of chemokine
levels, may contribute to a treated cell's resistance to neoplasia or
carcinogenesis. In another embodiment, cell membrane stabilization may
ameliorate or prevent tissue injury arising in the course of an
intestinal disease. In another embodiment, anti-oxidant action may limit
oxidative damage to cell and blood components arising in the course of an
intestinal disease. In another embodiment, attenuation of chemokine
levels may attenuate physiological reactions to stress that arise in the
course of an intestinal disease.

[0240] In one embodiment of the invention, the compounds for use in the
present invention described herein can be used to treat disease, through
amelioration, or prevention, of tissue injury arising in the course of
pathological disease states by stabilizing cell membranes; limiting
oxidative damage to cell and blood components; or attenuating
physiological reactions to stress, as expressed in elevated chemokine
levels.

[0241] In another embodiment, methods of the present invention involve
treating a subject by inter alia controlling the expression, production,
and activity of phospholipases such as PLA2; controlling the production
and/or action of lipid mediators, such as eicosanoids, platelet
activating factor (PAF) and lyso-phospholipids; amelioration of damage to
cell surface glycosaminoglycans (GAG) and proteoglycans; controlling the
production of oxidants, oxygen radicals and nitric oxide; protection of
cells, tissues, and plasma lipoproteins from damaging agents, such as
reactive oxygen species (ROS) and phospholipases; controlling the
expression, production, and activity of cytokines, chemokines and
interleukins; anti-oxidant therapy; anti-endotoxin therapy or any
combination thereof.

[0242] In one embodiment of the invention, the term "controlling" refers
to inhibiting the production and action of the above mentioned factors in
order to maintain their activity at the normal basal level and suppress
their activation in pathological conditions.

[0243] It will be appreciated by one skilled in the art that the compounds
characterized by the structures (A), (I), (II), (III), (IV), (V), (VI),
(VII), (VII), (IX), (IXa), (IXb), (X), (XI), (XII), (XIII), (XIV), (XV),
(XVI), (XVII), (XVIII), (XIX), (XX), (XXI), (XXII), or any combination
thereof may be administered according to any regimen, at any dosage, to
suit a particular application, for example cancer type or cancer stage,
or a particular subject, for example, male versus female, or for example,
in consideration of the age and lifestyle choice of the subject. In some
embodiments, such varied regimens are a function of the presence of
preneoplastic lesions or frank neoplasia, or in some embodiments, the
occurrence of metastasis.

[0244] Without further elaboration, it is believed that one skilled in the
art can, using the preceding description, utilize the present invention
to its fullest extent. The following preferred specific embodiments are,
therefore, to be construed as merely illustrative, and not limitative of
the remainder of the disclosure in any way whatsoever.

[0247] Boyden chamber chemoinvasion assays were performed as previously
described (Reich, R., M. et al. Clin Exp Metastasis, 13, 134-40 (1995)).
Matrigel (reconstituted basement membrane; 25 microgram) was dried on a
polycarbonated filter (Nucleopore® Polyester PVP free; Whatman
International Ltd., UK). Fibroblast conditioned medium (obtained from
confluent NIH-3T3 cells cultured in serum free DMEM) is used as the
chemoattractant. Cells were harvested by brief exposure to 1 mM EDTA,
washed with DMEM with 5 microgram collagen IV instead of Matrigel. This
amount of collagen does not form a barrier to the migrating cells but
rather an attachment substratum, and thus serves to measure cell
motility.

Determination of MMP Activity (Zymography)

[0248] Sub-confluent cell cultures were incubated for 6/24 h in serum-free
DMEM and the resulted supernatant was analyzed for collagenolytic
activity. The collagenolytic activity was determined on a gelatin
impregnated (1 mg/ml, Difco, USA), SDS-PAGE 8% gel, as previously
described (Brassart, B., A. et al. Clin Exp Metastasis, 16, 489-500
(1998)). Containing 0.1% BSA, and added to the Boyden chambers (200,000
cells). The chambers were incubated at 37° C. in humidified
atmosphere of 5% CO2/95% air for 6 h. The cells have traversed the
Matrigel layer and attached to the lower surface of the filter and
stained with Diff Quick (Dade Diagnostics, USA) and counted in five
random fields. The mean of the counts was calculated and values are
expressed in terms non-treated HT-1080 cells normalized to 100%.

Determination of Cell Chemotaxis

[0249] To rule out the possibility that the used inhibitors affect cell
motility, chemotaxis evaluation was performed in a similar way to
basement membrane invasion, with the exception that the filters are
coated. The bands were scanned (Epson Perfection 3200 Photo), and the
intensity was determined with the NIH image 1.63 software. All values are
expressed in terms in of untreated HT-1080 cells divided by the
absorbance of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide viability assay (MTT) [Kudo, I. & M. Murakami. Prostaglandins
Other Lipid Mediat, 68-69, 3-58 (2002)] normalized to 100%.

Determination of Cell PLA2 Activity

[0250] Confluent HT-1080 cells were metabolically labeled with either
[3H-AA] or [3H-OA](0.5 microCi/24 well plate) (Amersham
Biosciences, UK), by overnight incubation with the radioactive fatty
acid, then washed and the temporal release of the labeled fatty acid to
the culture medium was monitored under the different treatments (Dan, P.,
et al. FEBS Lett, 383, 75-8 (1996).

examined. HT-1080 cells were incubated for 24 h with HyPE, then washed,
and challenged to cross through a Matrigel layer coated filter in a
Boyden chamber.

[0257] FIG. 1 demonstrates that pre-treatment of the cancer cells with
HyPE effectively inhibited cell invasiveness without affecting cell
viability or motility (not shown). It should be emphasized that cells
were treated with HyPE prior to interaction with Matrigel and no ExPLI
(lipid conjugates) was added during the invasion assay. In addition, as
shown in FIG. 1, hyaluronic acid (HA) alone (without the lipidic portion
of the ExPLI) did not affect cell invasiveness, demonstrating that the
reduced invasiveness of cells after HyPE treatment is not due to
ExPLI-exerted steric hindrance between the cells and the Matrigel.

[0258] Since the invasion of the basement membrane is dependent on the
presence of collagen type IV degrading enzymes, the effect of HyPE effect
on MMP-2 and MMP-9 secretion by the tumor cells was evaluated. Culture
medium of HyPE-treated HT-1080 cells was collected and its collagenolytic
activity was determined. FIG. 2 shows that the collagenolytic activity of
both enzymes in the medium of HyPE-treated cells was reduced as a
function of PLA2 inhibitor concentration. Here too, treatment of
cells with the GAG moiety alone did not inhibit MMP production.

[0259] The following Table demonstrate ED (concentration that exert 50%
inhibition) of production of MMP-9 and MMP-2 by Human Fibrosarcoma
(HT-1080) cells.

Example 2

ExPLI (Lipid Conjugates) Effects on PLA2 Activity

[0260] The direct effect of HyPE on PLA2 activity in HT-1080 cells
was determined. Since cPLA2 is specific to AA-carrying
phospholipids, while sPLA2 has no fatty acid preference, the cell
membrane phospholipids were metabolically pre-labeled with either
radioactively-labeled AA or oleic acid (OA), and the temporal fatty acid
secretion to the culture medium was determined. FIG. 3 demonstrates that
treatment of HT-1080 cells with an ExPLI (lipid conjugates) inhibited the
release of both AA and OA.

[0261] These findings together suggest that both sPLA2 and cPLA2
are involved in these processes, but since both activities are inhibited
by the cell-impermeable inhibitor, it appears that they are controlled by
sPLA2.

[0262] Examination of the time course of the fatty acid release depicted
in FIG. 3 shows that at 1 h, OA production, catalyzed by sPLA2, is
higher than that of AA, while the reverse is observed at 2 h. In
addition, AA production is significantly enhanced at 2 h, while that of
OA is relatively higher at 1 h. Moreover, at both time points, treatment
with sPLA2 inhibitor suppressed AA production to the level of the
control, untreated cells. This may suggest that in HT-1080 cells the
activity of sPLA2 (producing both OA and AA) precedes that of
cPLA2 (producing only AA), and raises the possibility that
cPLA2 is activated subsequent to sPLA2 action.

[0263] As noted above, sPLA2 may act as a lipolytic enzyme and/or as
a receptor ligand. RT-PCR was used to determine sPLA2 types that are
expressed in the HT-1080 cells. Two receptor-ligand sPLA2s reported
to act via M-type receptors, specifically IB and X, and two sPLA2s
that act mainly as lipolytic enzymes, specifically HA and V were
investigated. Human HT-1080 fibrosarcoma cells express sPLA2-IB,
sPLA2-IIA and sPLA2-V as shown in FIG. 4. The cytosolic
cPLA2-IV alpha was identified as well. FIG. 4 also shows that
HT-1080 cells express the receptor to sPLA2-IB, thus implying the
presence of all the components required for a PLA2-mediated cell
signaling.

[0264] Exogenous sPLA2 may act as a lipolytic enzyme, hydrolyzing
cell membrane phospholipids, and also as receptor ligand, independent of
its lipolytic activity. Both these activities may lead to cPLA2
activation, as sPLA2-produced lyso-phospholipids and
receptor-mediated cell signaling lead to cPLA2 phosphorylation,
which is required for its activation. To differentiate between the two
potential mechanisms for the activation of MMP production, exogenous
sPLA2s were subjected to boiling, which is expected to inactivate
their lipolytic activity, and MMP production by HT-1080 was determined
following treatment with the native and boiled sPLA2s. Two
commercially-available sPLA2s were employed; porcine pancreatic
(Type-IB), for which HT-1080 cells express a receptor, and Crotalos atrox
(Type-HA) for which HT-1080 cells has no receptor. As shown in FIG. 5,
boiling considerably suppressed the lipolytic activity of Type-IIA
PLA2, but had a small inhibitory effect on that of Type-IB (about
20%). On the other hand, production of both MMP-2 and MMP-9 was elevated
by Type-IB PLA2, in a concentration-dependent manner, as shown in
FIG. 6. However, heating impaired the enzyme-receptor recognition, as
heat-inactivation of sPLA2-IB considerably suppressed its capacity
to induce MMP production (to a much larger extent than the boiling effect
on its lipolytic activity). MMP production was not affected by
sPLA2-IIA (not shown), nor was it attenuated by its heat
inactivation, which inhibited its lipolytic activity (FIG. 5). These
findings suggest that the induction of MMP production by sPLA2 is
mainly by a receptor-mediated process, rather than phospholipid
hydrolysis-dependent

[0265] The present study supports findings that AA-derived eicosanoids are
required for MMP production by the concomitant production of MMP and AA
and inhibition of MMP secretion by the ExPLI (FIGS. 2 and 3). Since
sPLA2-dependent lipolysis does not contribute significantly to MMP
production, one would assume that the required AA is provided by
cPLA2. This enzyme can be activated by phosphorylation that is
induced by sPLA2 receptor-mediated signaling, as has been previously
reported for IB-sPLA2. To examine this possibility in the present
system, the phosphorylation status of cPLA2 by native and
heat-inactivated types IB and IIA sPLA2 was assessed, and its
inhibition by ExPLI (lipid conjugates). As shown in FIG. 7, sPLA2-IB
strongly enhanced cPLA2 phosphorylation, and this was reduced to the
basal level by heat inactivation of the enzyme or treatment with ExPLI
(lipid conjugates). At the same time, sPLA2-IA did not lead to any
cPLA2 phosphorylation (not shown).

[0266] To further elaborate on the specific involvement of IB-PLA2 in
induction of MMP production, the ExPLI (lipid conjugates) effect on PLA2
mRNA expression was determined, using RT-PCR. As shown in FIG. 8,
treatment of HT-1080 cells with ExPLI (lipid conjugates) had no effect of
IIA-PLA2 expression, but considerably reduced (by 50%) the
expression of PLA2-IB, concomitantly with the above shown inhibition
of cell invasiveness (FIG. 1), MMP production (FIG. 2) and cPLA2
phosphorylation (FIG. 6).

Example 3

Invasive Cellular Proliferative Disorders

[0267] The process of cancer spread entails multiple events, each of these
is a worthy target for inhibitory drug action, including the rate of
cell-proliferation, the rate of spread through blood vessels, the rate of
invasiveness through contiguous and non-contiguous (metastases) tissues,
and the rate of production of new blood vessels to supply the cancerous
growth. Cancer cells frequently produce intracellular matrix tissue
degrading enzymes which serve to enhance their invasive potential. Cancer
is thus a multiphasic disease involving the process of tissue
invasiveness, spread through tissue channels, angiogenesis and tumor
vascularization. These latter processes depend upon the rates of
proliferation of endothelial cells and smooth muscle cells.

[0268] Lipid-conjugates inhibit the production and activities of enzyme
that break the basal membrane and enable the invasion of cancer cells,
such as collagenase (metaloproteinase=MMP), heparinase and hyaluronidase:

[0269] To demonstrate the Lipid-conjugate effect on collagenase, HT-1080
(fibrosarcoma) cells were incubated for 24 h with HYPE at the indicated
concentration. The culture medium was then collected and its collagenase
activity was determined by a zymographic assay. Each datum is average of
two plates (FIG. 11).

[0270] To demonstrate the ability of the Lipid-conjugates to inhibit
hyaluronidase activity, hyaluronic acid (HA) in PBS (0.75 mg/ml) was
interacted with hyaluronidase (15 U/ml) in the absence or presence of
HYPE, at the indicated concentration for 1 h. HA degradation was
determined by the change in the viscosity of its solution (FIG. 12).

[0271] To demonstrate the inhibition of heparinase activity by
Lipid-conjugates, BGM cells were incubated overnight with 50 μCi
35SO42- per well (to label the cell surface
glycosaminoglycans). The cells then were washed 3 times with PBS before
treating with 5 units of heparinase I in 200 μl PBS for 3 h. The
medium was collected and its 35S content was counted (FIG. 13).
Recombinant heparanase enzyme, in the absence or presence of the lipid
conjugates HyPE -or CSAPE) was incubated for 16 h (37° C., pH 6.2)
on dishes coated with sulfate-labeled ECM, prepared as described
(Vlodavsky et al., Cancer Res 43:2704-2711, 1983). Sulfate-labeled
material released into the incubation medium was analyzed by gel
filtration on a Sepharose 6B column. Nearly intact heparan sulfate
proteoglycans are eluted just after the void volume (peak I, Kav<0.2,
fractions 1-10) and heparan sulfate degradation fragments are eluted
later with 0.5<Kav<0.8 (peak II, fractions 15-35). These fragments
were shown to be degradation products of HS as they were 5-6 fold smaller
than intact HS side chains, resistant to further digestion with papain
and chondroitinase ABC, and susceptible to deamination by nitrous acid.

[0272] For showing the ability of the Lipid-conjugates to inhibit the
invasion of tumor cells through basement membrane, the chemoattractant
invasion assay was used: Polycarbonate fibers, 8 μm pore size, were
coated with 25 μg of a mixture of basement membrane components
(Matrigel) and placed in modified Boyden chambers. The cells
(2×105) were released from their culture dishes by a short
exposure to EDTA (1 mM), centrifuged, re-suspended in 0.1% BSA/DMEM, and
placed in the upper compartment of the Boyden chamber. Fibroblast
conditioned medium was placed in the lower compartment as a source of
chemoattractants. After incubation for 6 h at 37 C, the cells on the
lower surface of the filter were stained with Diff-Quick (American
Scientific Products) and were quantitated with an image analyzer (Optomax
V) attached to an Olympus CK2 microscope. The data are expressed relative
to the area occupied by untreated cells on the lower surface of the
filter. (Albini et al., A Rapid In Vitro Assay for Quantitating the
Invasive Potential of Tumor Cells. Cancer Res. 47:3239-3245, 1987). FIG.
10A demonstrates the Lipid-conjugate ability to attenuate cancer cell
invasiveness.

[0273] Further experiments utilizing a Boyden chamber for chemo-invasion
assays were performed: Matrigel (25 ug) was dried on a polycarbonate
filter (PVP-free, Nucleopore). Fibroblast-conditioned medium (obtained
from confluent NIH-3T3 cells cultured in serum-free DMEM) was used as the
chemo-attractant. HT-1080 human fibrosarcoma cells were harvested (by
brief exposure to 1 mM EDTA), washed with DMEM containing 0.1% bovine
serum albumin, and added to the Boyden chamber (200k cells). The chambers
were incubated in a humidified incubator at 37° C. (5% CO2
95% air) for 6 h. The cells that have traversed the Matrigel layer and
attached to the lower surface of the filter were stained with Diff Quick
(American Scientific Products) and counted. The results presented in FIG.
10B clearly demonstrated the inhibitory effect of dipalmitoyl
phosphatidylethanolamine hyaluronic acid (HyPE) and dimyristoyl
phosphatidylethanolamine hyurolonic acid (HyDMPE) indicate the actual
compounds (FIG. 10).

[0274] For demonstrating Lipid-conjugate effect on proliferation of
endothelial cells, bovine aortic endothelial cells were plated in culture
dishes for 6 h, then washed to remove unattached cells. The remaining
attached cells were incubated in the absence (control) or presence of
Lipid-conjugates at the indicated concentration, and stimulated with VEGF
(vascular endothelial growth factor) for 48 h. The cells were then
washed, collected by trypsinization and counted in a Coulter counter. The
results are mean±S.D. for 3 replications. *p<0.005 (FIG. 10C).

[0276] The capacity of the lipid-conjugates to control angiogenesis is
illustrated in FIG. 10E. This Figure demonstrates the inhibitory effect
induced by HyPE on capillary tube formation by HBMEC, in a
three-dimensional fibrin gel, stimulated by the above growth factors.
HyPE (20 μM) or hyaluronic acid (the carrier without the lipid moiety)
were added to the HBMEC-coated beads in the fibrin simultaneously with
the growth factors. Line A: control, Line B: b-FGF (25 ng/ml), Line C:
VEGF (20 ng/ml), Line D: OSM (2.5 nm/ml). Column 1: Without HyPE, Column
2: HyPE 20 μM, Column 3: Hyaluronic acid 20 μM.

[0277] This raises the possibility that the observed inhibitory effect
might be due to interference of the polymeric carrier with the
accessibility of the growth factors to the cell surface. To examine this
possibility, HBMEC cultured on the microcarrier beads were first
stimulated with the growth factors for 3 h (to allow interaction with
their receptors at the cell surface), then washed to remove the unbound
growth factors and introduced into HyPE-containing fibrin matrix. As
shown in FIG. 10F, under these conditions, capillary tube formation was
effectively suppressed by HyPE, suggesting that the HyPE effect is not
due to a defective growth factor accessibility due to steric hindrance by
the polymer at the cell surface of the endothelial cells. Line A: b-FGF
(25 ng/ml), Line B: VEGF (20 ng/ml), Line C: OSM (2.5 nm/ml). Column 1:
Without HyPE, Column 2: HyPE 20 μM.

[0278] HyPE inhibits bFGF-, VEGF- and OSM-stimulated Capillary Tube
Formation in a three-dimensional fibrin Gel. The corresponding
quantitation of the capillary formation is presented in the following
Table:

[0279] Bovine aortic endothelial cells were seeded in the absence and
presence of HyPE or HyDMPE at the indicated concentration, on a layer of
Matrigel (in culture dishes), enabling 3-dimensional growth and formation
of capillaries. The capillary length was determined, using image analysis
program, after 5 hours. The following Table demonstration the inhibition
of capillary formation (angiogenesis) by lipid conjugates. Data are
expressed as % of control (untreated):

[0280] In addition, the anti-proliferative effects of the Lipid-conjugates
on bovine aortic smooth muscle cells, unstimulated or stimulated by
thrombin, and on the proliferation of human venous smooth muscle cells
was demonstrated:

[0281] For unstimulated cells, bovine aortic smooth muscle cells were
seeded at 7×103 cells per well (in 24-well plates), in DMEM
supplemented with 10% FCS, in the absence or presence of HYPE-40 or
HYPE-80 (enriched with PE), grown for 72 h, and counted in Coulter (FIG.
14).

[0282] For stimulated cells, bovine aortic smooth muscle cells were grown
under the conditions as above for 48 h, following pre-incubation for 6 h,
as indicated, with either thrombin, fetal calf serum, Lipid-conjugate, or
both. Cell growth is represented as the amount of thymidine incorporation
(FIG. 15).

[0283] Smooth muscle cells (SMC) from human saphenous vein, were
inoculated at 8×10'/cells/5 mm culture dish, in DMEM supplemented
with 5% fetal calf serum and 5% human serum. A day later the cells were
washed and incubated in the same culture medium in the absence (control)
or presence of the Lipid-conjugate (HEPPE) or its polymeric carrier
(heparin, at the same concentration as the HEPPE). After 5 days the cells
were harvested (by trypsinization) and counted (FIG. 13). Each datum is
mean±SEM for 3 replications (the same results were obtained in a
second reproducible experiment). *p<0.005.

[0284] Effect of Lipid-conjugates on mouse lung metastases formation
induced by mouse melanoma cells: 105 B16 F10 mouse melanoma cells were
injected I.V. into a mouse (20-25 g). Three weeks later the lungs were
collected and the metastases on the lung surface counted. The
Lipid-conjugate effect, illustrated in FIG. 10G, was examined as follows:
In experiment I, the indicated Lipid-conjugate (HyPE, CSAPE, HemPE) was
injected I.P. (1 mg/mouse) 5 times a week for 3 weeks starting on day 1
(total of 15 injections) (FIG. 10G-I).

[0285] In FIG. 10G-II, HYPE (selected subsequently to experiment I) was
injected I.P. (1 mg/mouse) as follows: A. 5 times a week for 3 weeks
starting on day 1 (total of 15 injections); B. 5 times a week for 2 weeks
starting from week 2 (total of 10 injections); C. One injection (I.P.)
simultaneously with I.V. injection of the melanoma cells. D=Mice injected
(I.P.) with hyaluronic acid alone (without PE), 5 times a week for 3
weeks, starting on day 1 (total of 15 injections). Each group included 6
mice. *p<0.0001, **p<1.10-5, ***p<2.10-7. The results clearly
demonstrate that the Lipid conjugates inhibit melanoma-induced lung
metastases.

[0286] These results support the notion that the Lipid-conjugates control
the proliferation of smooth muscle cells, which is essential for tumor
vascularization subsequent to capillary formation by endothelial cells.

[0287] Taken together, the experiments described above, demonstrate that
administration of the Lipid-conjugates are effective therapy in the
treatment of cancer growth and metastasis, by a plurality of mechanisms,
including suppression of cell proliferation, invasion of cancer cells,
angiogenesis and metastasis formation and tumor vascularization.

[0288] Thus, Lipid-conjugates are effective therapy for cellular
proliferative disorders, such as cancer. The process of cancer spread
entails multiple events, each of these is a worthy target for inhibitory
drug action, including the rate of cell-proliferation, the rate of spread
through blood vessels, the rate of invasiveness through contiguous and
non-contiguous (metastases) tissues, and the rate of production of new
blood vessels to supply the cancerous growth. Cancer cells frequently
produce intracellular matrix tissue degrading enzymes which serve to
enhance their invasive potential. Cancer is thus a multiphasic disease
involving the process of tissue invasiveness, spread through tissue
channels, angiogenesis and tumor vascularization. These latter processes
depend upon the rates of proliferation of endothelial cells and smooth
muscle cells.

[0289] Three types of sPLA2s are expressed in HT-1080 cells: IB, IIA
and V. These cells also express the M-type sPLA2 receptor. These
enzymes differ in their mode of action. IB exhibits low catalytic
activity along with independent high affinity for M-type sPLA2
receptor. The receptor-mediated signaling reportedly leads to activation
of cPLA2, which is a major source of cellular AA release. The IIA
and V are structurally close heparin-binding isoforms participating in
stimulus-induced AA release. In the present study we employed exogenous
enzymes that represent the two sPLA2 types, namely porcine
pancreatic-derived (Type IB) and crotalos atrox venom-derived (Type IIA)
forms, to differentiate between the lipolytic and receptor-mediated
contributions to MMP production and cell invasiveness.

[0290] The results presented herein show that MMP-2/9 production by human
fibrosarcoma HT-1080 cells and their invasiveness (FIGS. 1 and 2)
correspond to AA production (FIG. 3), and these activities are
concomitantly inhibited by the cell-impermeable sPLA2 inhibitor
(ExPLI). It further shows that sPLA2-IB activates MMP production
(FIG. 8) via a receptor-mediated process, rather than its lipolytic
activity (FIGS. 5 and 6). Concomitantly, sPLA2-IB activates
cPLA2 by its phosphorylation (FIG. 7), and intracellular cPLA2
phosphorylation is induced by M-type sPLA2 receptor interaction. All
the above processes are inhibited by the ExPLI (lipid conjugates), thus
assigning a pivotal role for sPLA2-IB in MMP activation and subsequent
cancer cell invasiveness. Taken together, these findings suggest that
sPLA2-IB-mediated MMP activation is compatible with the sequence of
events illustrated in FIG. 9: sPLA2-IB secreted to the extracellular
medium interacts with its membrane receptor (on its own and neighboring
cells), signals the phosphorylation and subsequent activation of the
cytosolic cPLA2, which provides the AA for production of the
eicosanoids required for MMP production/action.

[0291] Of specific interest is the finding that although sPLA2-IB
induces MMP production by acting as a receptor-ligand, rather by its
lipolytic activity, its effect is suppressed by the ExPLI (lipid
conjugates), which is designed to inhibit membrane phospholipids
hydrolysis. Additionally, it was found that in parallel to inhibition of
MMP production, the ExPLI (lipid conjugates) reduced the production of
AA, which attributed to cPLA2, and also OA, which is a product of
sPLA2 and other PLA (but not cPLA2). It is thus possible that
lipolytic activity of sPLA2 and/or PLA2 also take part in
sPLA2-IB-induced MMP production. The results indicate that activated
cPLA2 provides the AA for production of eicosanoids required for MMP
activation/action.

Example 5

Toxicity Tests

[0292] The following compounds were tested: HyPE, CMPE, CSAPE and HepPE.
The compounds were injected IP at one dose of 1000, 500 or 200 mg/Kg body
weight. Toxicity was evaluated after one week, by mortality, body weight,
hematocrit, blood count (red and white cells), and visual examination of
internal organs after sacrifice. These were compared to control,
untreated mice. Each dose was applied to a group of three mice. No
significant change in the above criteria was induced by treatment with
these compounds, except for the HepPE, which induced hemorrhage.

[0293] The non-toxicity of the Lipid conjugates is demonstrated in Table 6
and Table 7, depicting the results obtained for HyPE in acute (6) and
long-term (7) toxicity tests.

[0294] For long-term toxicity test of HyPE, a group of 6 mice received a
dose of 100 mg HyPE/Kg body weight, injected IP 3 times a week for 30
weeks (total of 180 mg to a mouse of 20 g). Toxicity was evaluated as for
Table 5. No mortality, and no significant change in the above criteria
was induced by this treatment, compared to normal untreated mice (see
Table 6), as depicted in Table 7.

[0295] 4 g of chlorocresol was dissolved in 4 L of deionized (DI) water
(0.1% o solution). HA UL 15 was dissolved in 4 L of 0.1% chlorocresol
solution with mechanical stirring. To prevent clogging of the
ultrafiltration membranes, the HA solution was filtered through a 100
μm filter followed by a 50 μm filter followed by a 10 μm filter,
all previously disinfected with 10% hydrogen peroxide and washed with
copious amounts of DI water to ensure hydrogen peroxide has been removed
(verified with peroxide-detecting strips).

Example 7

Ultrafiltration Fractionation of Hyaluronic Acid (HA)

[0296] HA solution of Example 6 was loaded into the Centramate system,
previously disinfected with 10% hydrogen peroxide and washed with copious
amounts of DI water to ensure hydrogen peroxide has been removed
(verified with peroxide-detecting strips).

[0297] By means of constant volume diafiltration with 70 kDa Omega TFF
membranes, 20 L of 0.1% chlorocresol solution, prepared as described in
Example 6, was ultrafiltered, collecting the filtrate, the fraction less
than 70 kDa, in a carboy, previously disinfected with 10% hydrogen
peroxide. The pump speed and valves shall be set such that the retentate
flow is ten times the filtrate flow and the feed pressure is less than 40
PSI.

[0298] The 70 kDa membranes were replaced with 30 kDa membranes and the
Centramate system was disinfected with 10% hydrogen peroxide.

[0299] 5 L of the filtrate, the fraction less than 70 kDa, were loaded
into the reservoir and by means of constant volume diafiltration, the
remaining 35 L in the carboys of the fraction less than 70 kDa were
ultrafiltered. The reservoir volume was reduced to 2 L and an additional
10 L of DI water was ultrafiltered to remove the chlorocresol (confirmed
by appropriate GC assay). The reservoir volume was further reduced to 1
L, reducing the pump speed, if necessary, to keep the feed pressure below
40 PSI. The reservoir was then emptied directly into an autoclaved
lyoguard container, closed, frozen and lyophilized to yield HA UF 70/30.
GPC analysis was performed to ensure that this lot of HA UF 70/30 was
consistent with earlier batches. A bioburden assay and an appropriate GC
assay for chlorocresol was performed. Karl Fischer analysis was performed
to determine the water content of HA UF 70/30.

Example 8

HyPE Synthesis Reaction

[0300] 24 g of 2-(N-morpholino)ethanesulfonic acid (MES) were dissolved in
125 mL of DI water and the pH was adjusted to pH 6.4 by addition of 4 N
NaOH.

[0301] 2.5 g of dipalmitoylphosphatidylethanolamine (DPPE) and 25 g of
hydroxybenzotriazole (HOBT) were dissolved in 940 mL of tert-butanol and
80 mL of water with stirring and heating at 45° C. in a 12 L round
bottom flask (forming a closed system with the pump and the sonciator,
all of which will have been previously autoclaved and/or disinfected with
70% isopropanol). To this was added 850 mL of water and 115 mL of the MES
solution. The pH of this solution was adjusted to pH 6.4 by addition of
2.5 N NaOH. 25 g of HA UF 70/30 of Example 7 were then dissolved with
stirring and heating at 45° C. 25 g of
1-ethyl-3-(3-dimethylaminoethyl)carbodiimide (EDAC) were then added, the
pump and the sonicator were turned on and the system was kept between 40
and 50° C. for 3 hours. GPC analysis was performed to monitor the
progress of the reaction. After 3 hours the sonicator and the pump were
turned off and the solution was stirred at room temperature overnight.
The following day 750 mL of acetonitrile were added to precipitate HyPE.
This was allowed to stand for 30 minutes after which the supernatant was
removed. To this was added 7.5 L of 2% Na2CO3, previously
prepared by dissolving 150 g of Na2CO3 in 7.5 L in DI water.
Vigorous mechanical stirring for at least 2 hours hydrolyzed urea related
byproducts. The solution was neutralized with 6 N HCl while the
temperature was kept at 20-25° C. by passing the solution through
a cooled, jacketed flow cell.

Example 9

Alkaline Ultrafiltration of HyPE

[0302] 2.25 kg of NaHCO3 was dissolved in 150 L of 0.1% chlorocresol
solution, prepared by dissolving 150 g of chlorocresol in 150 L of DI
water. By means of valves, the closed reaction system was diverted so
that the digested, neutralized HyPE solution of Example 8 was pumped from
the round bottom flask to the centrasette system. By means of constant
volume diafiltration with a 10 kDa Omega TFF membrane, 150 L of 1.5%
NaHCO3 in 0.1% chlorocresol solution was ultrafiltered, discarding
the filtrate, the fraction less than 10 kDa. The pump speed and valves
were set such that the retentate flow was ten times the filtrate flow and
the feed pressure was less than 40 PSI. GPC analysis was performed to
ensure the disappearance of urea-related peaks at ˜13.2 min and the
HOBT peak at ˜17.2 min. The solution was neutralized with 6 N HCl
while the temperature was kept at 20-25° C. by passing the
solution through a cooled, jacketed flow cell.

Example 10

Extraction of HyPE

[0303] An extraction solution was made by mixing 3 L of dichloromethane, 3
L of ethanol and 2.25 L of methanol. 7.5 L of the extraction solution was
added to a round bottom flask containing 3 L of crude HyPE solution of
Example 9. This was stirred vigorously for 15 minutes after which time it
was allowed to stand for 45 min. The lower dichloromethane layer was
removed. By means of constant volume diafiltration the solution was
washed with 100 L of DI water to remove the methanol and ethanol. GPC
analysis was performed to ensure the disappearance of peaks at ˜14
min. The volume was reduced to 3 L and emptied directly into 2 autoclaved
lyoguard containers, closed, frozen and lyophilized to yield HyPE. NMR
and HPLC data for isolated HyPE are shown in FIG. 16 and FIG. 17.

Example 11

Preparation of Hype from 9.54 kD Hyaluronic Acid

[0304] MES buffer was prepared by dissolving 14.5 g of MES in 75 mL of
DI-H2O and adjusting the pH to 6.4 with 4N NaOH. Using an apparatus
similar to that depicted in FIG. 18, 10.0 g of HOBT was dissolved in 225
mL of DI-H2O, 60 mL MES buffer, 12 mL of tert-butanol. The pH was
adjusted to 6.4 with 4N NaOH. 15.1 g of HA was dissolved in 350 mL of
DI-H2O. 1.25 g or DPPE was dissolved in 440 mL of tert-butanol and
90 mL DI-H2O with heating to 55 deg C. The solutions of HA and HOBT
were warmed to 35 deg C. and mixed. The DPPE solution, at 50 deg C. was
then added to afford a clear solution. This was allowed to cool to 43 deg
C., when it was added to the flask and circulated through the sonoreactor
system. Some component of the reaction mixture came out of solution and
it was necessary to heat the reaction mixture to 49 deg C. with
sonication to form a clear solution. 12.5 g of EDAC was added as a powder
to the reaction mixture at a temperature of 45 deg C. Sonication began
with a power of 180 watts. The reaction was monitored by GPC as shown in
FIG. 19 (after 6 h) and because the extent of agglomeration, as observed
by the ratio of the area of the first peak to that of the second
continued to increase, the reaction was allowed to continue beyond the
normal 3 h and was continued the next day. The sonication was turned off
and the reaction mixture was filtered through a 0.45 μm filter to
remove a small amount of rubber debris apparently from the stator. The
solution (1200 mL) was extracted with 600 mL DCM and 600 mL MeOH. The
resulting emulsion quickly resolved and the aqueous layer was extracted
again with 500 mL DCM and 500 mL EtOH. Finally, the aqueous layer was
extracted with 250 mL DCM and 250 mL EtOH and left over the weekend.
Residual DCM was removed by rotovaporation at 35 deg C. and 200 Torr. The
solution was then transferred to a previously cleaned centrasette
ultrafiltration system with a 10 kDa membrane and by constant volume
diafiltration was washed with 5 L of 1.5% NaHCO3 to remove residual
organic solvents. The pH was then increased by slow addition of 2%
Na2CO3 to pH 9.2. The solution was stirred for 1 hour at room
temperature. After further washing with 30 L of 1.5% NaHCO3 the peat
at -12.5 min had disappeared and the solution was washed with 30 L of
DI-H2O until pH 7. To remove any digestion/ultrafiltration
byproducts, such as free palmitic acid, the solution was then extracted
again with 1 L DCM, 1 L MeOH and 0.75 L EtOH. The aqueous layer was
extracted again with 400 mL DCM and 50 mL EtOH and finally a third time
with 400 mL DCM and 50 mL EtOH. Residual DCM was removed by
rotovaporation at 30 deg C. and 200 Torr. By constant volume
diafiltration residual MeOH and EtOH were removed by washing with 15 L
DI-H2O. The solution was concentrated to 1 L and filtered through a
0.2 μm filter into a lyoguard container and placed in the lypholizer.
It was frozen by lowering the shelf temperature to -70 deg C. When
frozen, vacuum was applied (14 mT) and the shelf temperature was raised
to 30 deg C. Five days later 6.134 g of HyPE was recovered with a
water-corrected weight of 5.2 g which corresponds to a 42% yield based on
12.5 g (water corrected) of HA. Total phosphorus was found to be 0.28%
(dry basis). By LC/MS assay, 1,456 ppm of free EDU were found and after
exposure to NaOH 12,557 ppm total EDU was found. No HOBT was detected and
MES was less than 80 ppm. GPC of the final product is shown in FIG. 20.

[0305] While certain features of the invention have been illustrated and
described herein, many modifications, substitutions, changes, and
equivalents will now occur to those of ordinary skill in the art. It is,
therefore, to be understood that the appended claims are intended to
cover all such modifications and changes as fall within the true spirit
of the invention.